Recombinant bacterium for synthesizing D-mannitol and application thereof

Abstract

The invention provides a recombinant bacterium for synthesizing D-mannitol, application of the recombinant bacterium and a method for synthesizing D-mannitol through biotransformation of a glucose raw material by using the bacterium. The recombinant strain obtains the capability of producing D-mannitol on the basis of knocking out pfkA and pfkB genes, and further obtains various strains with higher capability of producing D-mannitol through a series of modifications such as knocking out stA, inserting pntA, inserting pntB and the like. The engineering strain has the prospect of green, efficient and industrial production of D-mannitol by utilizing the capability of synthesizing D-mannitol by glucose.

Description

Technical Field

[0001] The present invention belongs to the field of synthetic biology and gene editing, and specifically relates to the synthesis of D-mannitol by industrial microorganisms edited by gene recombination. Background Art

[0002] D-mannitol is a rare hexitol, which mainly exists in vegetables, fruits, fungi, bacteria and other microorganisms in nature. D-mannitol is a white or colorless crystalline powder, odorless, with a refreshing sweet taste, about 70% of the sweetness of sucrose, and a caloric value of only 1.99kcal / g. Due to its low calorie and low dental caries characteristics, it can be used as a sweetener and stabilizer in the food industry. The blood sugar and insulin index of D-mannitol is zero. As a functional food, it can be absorbed and utilized by intestinal microbial flora, thereby achieving the effect of lowering blood lipids and losing weight. In 1972, D-mannitol-related products have passed the GRAS (Generally Recognized As Safe) certification of the US Food and Drug Administration (FDA). In addition, D-mannitol is also a prerequisite molecule for some chemically synthesized drugs. In short, D-mannitol has great application value in industries such as food additives, health products and pharmaceuticals.

[0003] There are three main methods for traditionally producing D-mannitol: plant extraction, chemical synthesis, and microbial transformation. Kelp extraction is the main method for producing mannitol in my country, which involves steps such as hydrolysis, filtration, separation, and purification. It has problems such as low extraction rate and high production cost. The catalytic reduction method is to hydrolyze sucrose to generate a mixture of glucose and fructose, and then catalyze hydrogenation under high temperature and strong acid conditions to generate a mixture of sorbitol and mannitol, and then obtain pure D-mannitol through a separation process. This method has harsh reaction conditions, requires metal catalysts, and has the problem of by-product accumulation. Summary of the invention

[0004] Due to the low yield of the extraction method and the high pollution of the chemical method, it is urgent to develop an effective biosynthesis method for D-mannitol.

[0005] The technical problem to be solved by the present invention is to provide a stable strain, which utilizes raw materials such as glucose, glycerol, sucrose, and fructose syrup to supply microbial growth and prepares D-mannitol using substrates such as glucose, fructose, sucrose, and fructose syrup.

[0006] In order to solve the above technical problems, the present invention provides a method for preparing D-mannitol, which comprises using the above recombinant Escherichia coli to prepare D-mannitol with substrates such as glucose, fructose, sucrose, and fructose syrup.

[0007] According to an exemplary embodiment of the present disclosure, the present disclosure provides a D-mannitol synthesis gene cluster, comprising:

[0008] Pyridine nucleotide transhydrogenase α subunit pntA gene or its isozyme gene or its functional homolog gene; and

[0009] Pyridine nucleotide transhydrogenase β subunit pntB or its isozyme gene or its functional homolog gene.

[0010] Optionally or alternatively, the gene cluster according to any of the preceding items further comprises:

[0011] 6-phosphate glucose dehydrogenase zwf gene or its isozyme gene or its functional homolog gene;

[0012] 6-phosphogluconate dehydrogenase gnd gene or its isozyme gene or its functional homolog gene; and

[0013] p119 promoter.

[0014] Optionally or alternatively, the gene cluster according to any of the preceding items further comprises:

[0015] Mannitol 1-phosphate-5-dehydrogenase M1PDH gene or its isozyme gene or its functional homolog gene; and

[0016] Mannitol-1-phosphatase M1PP gene or its isozyme gene or its functional homolog gene.

[0017] Optionally or alternatively, according to any of the preceding gene clusters, wherein,

[0018] 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).

[0019] Optionally or alternatively, according to any of the preceding gene clusters, wherein,

[0020] The M1PDH gene comprises or consists of a sequence shown in any one of SEQ ID NOs. 1-9.

[0021] Optionally or alternatively, according to any of the preceding gene clusters, wherein,

[0022] 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 Escherichiacoli (ECM1PPA, ECM1PPB).

[0023] Optionally or alternatively, according to any of the preceding gene clusters, wherein,

[0024] The M1PP gene comprises or consists of a sequence shown in any one of SEQ ID NOs. 10-12.

[0025] Optionally or alternatively, according to any of the preceding gene clusters, wherein,

[0026] pntA and pntB contain the sequences shown in Gene ID: 946628 and Gene ID: 946144, respectively;

[0027] Optionally, the zwf gene comprises the sequence shown in Gene ID: 946370;

[0028] Optionally, the gnd gene comprises the sequence shown in Gene ID:946554.

[0029] In an exemplary embodiment of the present disclosure, the present disclosure provides a use of the gene cluster according to any one of the foregoing items in producing D-mannitol.

[0030] Optionally or alternatively, according to any of the aforementioned uses, the use is to produce D-mannitol by fermentation of engineered cells, preferably, the engineered cells are genetically recombinant Escherichia coli.

[0031] Optionally or alternatively, according to any of the preceding claims, wherein in the engineered cell:

[0032] The expression of 6-phosphofructokinase 1pfkA gene or its isozyme gene or its functional homolog gene is inhibited, and / or the activity of the protein, domain or functional fragment encoded by the pfkA gene or its isozyme gene is inhibited; and

[0033] The expression of 6-phosphofructokinase 2pfkB gene or its isozyme gene or its functional homolog gene is inhibited, and / or the activity of the protein, domain or functional fragment encoded by the pfkB gene or its isozyme gene is inhibited.

[0034] Optionally or alternatively, according to any of the preceding claims, wherein in the engineered cell:

[0035] The expression of the pyridine nucleotide transhydrogenase sthA gene or its isozyme gene or its functional homolog gene is inhibited, and / or the activity of the protein, domain or functional fragment encoded by the sthA gene or its isozyme gene is inhibited.

[0036] Optionally or alternatively, according to any of the preceding claims, wherein in the engineered cell:

[0037] The expression of lauroyl acyltransferase LPXL gene, its isozyme gene or its functional homolog gene is inhibited, and / or the activity of the protein, domain or functional fragment encoded by the LPXL gene or its isozyme gene is inhibited.

[0038] Optionally or alternatively, according to any of the preceding claims, wherein in the engineered cell:

[0039] The expression of the phosphogluconate dehydratase edd gene or its isozyme gene or its functional homolog gene is inhibited, and / or the activity of the protein, domain or functional fragment encoded by the edd gene or its isozyme gene is inhibited; and

[0040] The expression of KHG / KDPG aldolase eda gene or its isozyme gene or its functional homolog gene is inhibited, and / or the activity of the protein, domain or functional fragment encoded by the eda gene or its isozyme gene is inhibited.

[0041] Optionally or alternatively, according to any of the preceding claims, wherein in the engineered cell:

[0042] The expression of the myristoyl transferase LPXM gene or its isozyme gene or its functional homolog gene is inhibited, and / or the activity of the protein, domain or functional fragment encoded by the LPXM gene or its isozyme gene is inhibited.

[0043] In an exemplary embodiment of the present disclosure, a genetically recombinant engineered cell is provided, wherein the engineered cell contains the following modifications:

[0044] 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

[0045] 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.

[0046] Optionally or alternatively, the engineered cell according to any of the foregoing items further comprises the following modifications:

[0047] 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;

[0048] 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

[0049] Introducing and / or enhancing the expression of the pyridine nucleotide transhydrogenase β subunit pntB gene or its isozyme gene or its functional homolog gene.

[0050] Optionally or alternatively, the engineered cell according to any of the foregoing items further comprises the following modifications:

[0051] 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;

[0052] Introducing and / or enhancing the expression of 6-phosphate glucose dehydrogenase zwf gene or its isozyme gene or its functional homolog gene;

[0053] Introducing and / or enhancing the expression of 6-phosphogluconate dehydrogenase gnd gene or its isozyme gene or its functional homolog gene; and

[0054] p119 promoter.

[0055] Optionally or alternatively, the engineered cell according to any of the foregoing items further comprises the following modifications:

[0056] 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

[0057] The KHG / KDPG aldolase eda gene or its isozyme gene or its functional homolog gene is deleted, has a nonsense mutation, has an inactivation mutation, or has an expression-inhibiting nucleic acid fragment.

[0058] Optionally or alternatively, the engineered cell according to any of the foregoing items further comprises the following modifications:

[0059] Myristoyltransferase LPXM gene or its isozyme gene or its functional homolog gene deletion, nonsense mutation, inactivation mutation or expression inhibitory nucleic acid fragment;

[0060] Introducing and / or enhancing the expression of mannitol 1-phosphate-5-dehydrogenase M1PDH gene or its isozyme gene or its functional homolog gene; and

[0061] Introducing and / or enhancing the expression of mannitol-1-phosphatase M1PP gene or its isozyme gene or its functional homolog gene.

[0062] Optionally or alternatively, according to any of the preceding engineered cells, wherein the M1PDH gene is from at least one of Thermoanaerobacter sp. (TSM1PDH), actococcus lactis subsp. lactisbv. 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).

[0063] Optionally or alternatively, according to any of the aforementioned engineered cells, the M1PDH gene comprises or consists of a sequence shown in any one of SEQ ID NOs. 1-9.

[0064] Optionally or alternatively, according to any of the preceding engineered cells, wherein 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), Escherichia coli (ECM1PPA, ECM1PPB).

[0065] Optionally or alternatively, according to any of the aforementioned engineered cells, the M1PP gene comprises or consists of a sequence shown in any one of SEQ ID NOs. 10-12.

[0066] Optionally or alternatively, according to any of the foregoing engineered cells, wherein pntA and pntB comprise the sequences shown in Gene ID: 946628 and Gene ID: 946144, respectively;

[0067] Optionally, the zwf gene comprises the sequence shown in Gene ID: 946370;

[0068] Optionally, the gnd gene comprises the sequence shown in Gene ID:946554.

[0069] Optionally or alternatively, according to any of the aforementioned engineered cells, it is Escherichia coli.

[0070] In an exemplary embodiment of the present disclosure, a method for producing D-mannitol is provided, comprising using any of the aforementioned engineered cells to ferment and produce D-mannitol.

[0071] Optionally or alternatively, the method according to any of the preceding claims further comprises synthesizing D-mannitol using glucose as a substrate.

[0072] In an exemplary embodiment of the present disclosure, there is provided use of the engineered cell according to any one of the foregoing items in producing D-mannitol.

[0073] The beneficial technical effects achieved by the present invention are as follows:

[0074] Based on the above-mentioned large-scale production problem of D-mannitol, the present invention develops a new D-mannitol production route based on a microbial fermentation method. The method uses cheap glucose and the like as production raw materials and utilizes microbial fermentation to synthesize high-purity D-mannitol in one step. The production process of D-mannitol using the present invention greatly shortens the production cycle, laying a solid foundation for its wide application and development.

[0075] The recombinant strain provided by the present invention can realize the growth of microorganisms using glucose, glycerol, sucrose, fructose syrup, etc. as carbon sources, and can also use mixed carbon sources containing the above carbon source components for growth. After the microorganisms grow to a certain biomass, glucose, fructose, sucrose, fructose syrup, etc. can be used as raw materials to synthesize D-mannitol. And then establish a corresponding technical route for synthesizing D-mannitol. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] 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.

[0077] Figure 1This 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.

[0078] Figure 2 This is the HPLC spectrum of D-mannitol standard.

[0079] Figure 3 HPLC spectrum of D-glucose standard.

[0080] Figure 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. DETAILED DESCRIPTION

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

[0110] 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%.

[0111] "Expression" refers to the transcription of a gene into structural RNA (rRNA, tRNA) or messenger RNA (mRNA), followed by translation into protein.

[0112] "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.

[0113] 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.

[0114] The term "pathway" or "metabolic pathway" is understood herein as a series of chemical reactions that build and break down molecules in a cell.

[0115] The nucleic acid sequence (ie, polynucleotide) or protein (ie, polypeptide) may be native or heterologous to the genome of the host cell.

[0116] "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.

[0117] 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.

[0118] 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.

[0119] As used herein, the term "vector" includes reference to autosomal expression vectors and integration vectors for integration into a chromosome.

[0120] 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.

[0121] "Plasmid" refers to an autonomously replicating extrachromosomal DNA that is not integrated into the genome of a microorganism and is usually circular in nature.

[0122] "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.

[0123] 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.

[0124] 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.

[0125] In the following examples, Escherichia coli MG1655 (CGSC#: 6300) was purchased from the Escherichia coli Genetic Collection Center of Yale University, USA.

[0126] 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).

[0127] 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).

[0128] 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).

[0129] 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).

[0130] 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).

[0131] 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).

[0132] 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).

[0133] 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).

[0134] 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)

[0135] 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).

[0136] 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).

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] Those skilled in the art will appreciate that other sequences include the above situations.

[0143] 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.

[0144] 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.

[0145] 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 fully described in the existing literature, 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, 2016; P. Nagarajan et al., Essentials of Laboratory Animal Science: Principles and Practices, Springer, 2021; and Jann Hau et al., Handbook of Laboratory Animal Science: Essential Principles and Practices, 4th edition, CRC Press, 2021.

[0146] The primer sequences in the following examples are as follows:

[0147] Table 1 Primer sequences

[0148]

[0149]

[0150]

[0151]

[0152] Example 1. Construction of recombinant Escherichia coli ML05

[0153] 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.

[0154] 1. Construction of strain ML01

[0155] 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.

[0156] The specific construction steps of the mutant ML01 of Escherichia coli K12 are as follows:

[0157] 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.

[0158] 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.

[0159] 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.

[0160] 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 A. By Figure 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.

[0161] 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.

[0162] 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 B. By Figure 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.

[0163] 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.

[0164] 2. Construction of strain ML02

[0165] ML02 is a product that uses CRISPER technology to replace the sthA gene of Escherichia coli K12 with

[0166] The recombinant bacteria are obtained by fragment pntAB of gene pntA and gene pntB, and other genes remain unchanged.

[0167] 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.

[0168] 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.

[0169] 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.

[0170] 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 C. By Figure 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.

[0171] 3. Construction of strain ML03

[0172] 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.

[0173] 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.

[0174] 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.

[0175] 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.

[0176] 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 D. By Figure 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.

[0177] IV. Construction of strain ML04

[0178] 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.

[0179] 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.

[0180] 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.

[0181] 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.

[0182] 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 E. By Figure 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.

[0183] 5. Construction of strain ML05

[0184] ML05 is a product that uses CRISPER technology to replace the gene lPXM of Escherichia coli K12 with

[0185] 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.

[0186] 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.

[0187] 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.

[0188] 2. Acquisition of Targeting Clips:

[0189] 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.

[0190] 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 F. By Figure 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.

[0191] Example 2. Construction of recombinant Escherichia coli ML06

[0192] 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.

[0193] 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.

[0194] 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.

[0195] Acquisition of target shooting clips:

[0196] 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.

[0197] 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 G. By Figure 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.

[0198] Abbreviated as ML06.

[0199] The genotypes of the Escherichia coli mutants in the embodiment are shown in Table 2:

[0200] Table 2 Genotypes of Escherichia coli mutants

[0201]

[0202] Example 3. Application of recombinant Escherichia coli ML05 in the preparation of D-mannitol using glucose as substrate

[0203] 1. Glycerol Growth Medium Composition

[0204] Growth medium components and final concentrations are as follows:

[0205] 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.

[0206] 2. Transformation medium composition

[0207] 25mM Na2HPO4, 25mM KH2PO4, 50mM MgCl2, 20% (g / 100mL) D-glucose, and the balance is water.

[0208] 3. Bacterial culture and enzyme induction

[0209] 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.

[0210] 4. Whole-cell catalysis of D-mannitol

[0211] 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.

[0212] D-mannitol standard was purchased from Shanghai McLean Biotechnology (Cat. No.: M813424) with a retention time of 16.838 min.

[0213] 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%.

[0214] 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%.

[0215] Table 3 D-mannitol production and conversion rate of various engineered bacteria using glucose as carbon source

[0216]

[0217] ML0* represents ML02 / ML03 / ML04 / ML05

[0218] Example 4. Application of recombinant Escherichia coli ML06 in the preparation of D-mannitol using fructose syrup as substrate

[0219] 1. Glucose growth medium composition

[0220] Growth medium components and final concentrations are as follows:

[0221] 25mM Na2HPO4, 25mM KH2PO4, 50mM NH4Cl, 5mM Na2SO4, 2mM MgSO4, 2% (g / 100mL) glucose, 0.5% (g / 100mL) yeast powder, trace elements: 50μM FeCl3, 20μMCaCl2, 10μM MnCl2, 10μMZnSO4, 2μM each of CoCl2, NiCl2, Na2MO4, Na2SeO3 and H3BO3, and the rest is water.

[0222] 2. Transformation medium composition

[0223] 25mM Na2HPO4, 25mM KH2PO4, 50mM MgCl2, 20% (g / 100mL) fructose syrup, and the balance is water.

[0224] 3. Bacterial culture and enzyme induction

[0225] 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.

[0226] 4. Whole-cell catalysis of D-mannitol

[0227] 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.

[0228] 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%.

[0229] Table 4 D-mannitol production and conversion rate of various engineered bacteria using fructose syrup as carbon source

[0230]

[0231] ML0* represents ML02 / ML03 / ML04 / ML05 / ML06

[0232] 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.

Claims

1. A D-mannitol synthesis gene cluster, comprising: Pyridine nucleotide transhydrogenase α subunit pntA gene or its isozyme gene or its functional homolog gene; and Pyridine nucleotide transhydrogenase β subunit pntB or its isozyme gene or its functional homolog gene.

2. The gene cluster according to any of the preceding claims, further comprising: 6-phosphate glucose dehydrogenase zwf gene or its isozyme gene or its functional homolog gene; 6-phosphogluconate dehydrogenase gnd gene or its isozyme gene or its functional homolog gene; as well as p119 promoter; Optionally, the gene cluster further comprises: Mannitol 1-phosphate-5-dehydrogenase M1PDH gene or its isozyme gene or its functional homolog gene; and 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).

3. A gene cluster according to any preceding claim, wherein: The M1PDH gene comprises or consists of a sequence shown in any one of SEQ ID NOs. 1 to 9; optionally, wherein, 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 Escherichiacoli (ECM1PPA, ECM1PPB); Optionally, wherein The M1PP gene comprises or consists of a sequence shown in any one of SEQ ID NOs. 10 to 12; Optionally, wherein pntA and pntB contain 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.

4. Use of the gene cluster according to any one of the preceding claims in the production of D-mannitol.

5. The use according to any of the preceding claims, wherein the use is the production of D-mannitol by fermentation of engineered cells, preferably, the engineered cells are genetically recombinant Escherichia coli; Optionally, wherein In the engineered cells: The expression of 6-phosphofructokinase 1pfkA gene or its isozyme gene or its functional homolog gene is inhibited, and / or the activity of the protein, domain or functional fragment encoded by the pfkA gene or its isozyme gene is inhibited; and The expression of 6-phosphofructokinase 2pfkB gene or its isozyme gene or its functional homolog gene is inhibited, and / or the activity of the protein, domain or functional fragment encoded by the pfkB gene or its isozyme gene is inhibited Optionally, wherein, in the engineered cell: The expression of the pyridine nucleotide transhydrogenase sthA gene or its isozyme gene or its functional homolog gene is inhibited, and / or the activity of the protein, domain or functional fragment encoded by the sthA gene or its isozyme gene is inhibited; Optionally, wherein, in the engineered cell: The expression of lauroyl acyltransferase LPXL gene or its isozyme gene or its functional homolog gene is inhibited, and / or the activity of the protein, domain or functional fragment encoded by the LPXL gene or its isozyme gene is inhibited; Optionally, wherein, in the engineered cell: The expression of the phosphogluconate dehydratase edd gene or its isozyme gene or its functional homolog gene is inhibited, and / or the activity of the protein, domain or functional fragment encoded by the edd gene or its isozyme gene is inhibited; and The expression of KHG / KDPG aldolase eda gene or its isozyme gene or its functional homolog gene is inhibited, and / or the activity of the protein, domain or functional fragment encoded by the eda gene or its isozyme gene is inhibited; Optionally, wherein, in the engineered cell: The expression of the myristoyl transferase LPXM gene or its isozyme gene or its functional homolog gene is inhibited, and / or the activity of the protein, domain or functional fragment encoded by the LPXM gene or its isozyme gene is inhibited.

6. A genetically recombinant engineered cell, the engineered cell comprising 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 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.

7. An engineered cell according to any preceding claim, further comprising 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.

8. An engineered cell according to any preceding claim, wherein The M1PP gene is from at least one of Pantoeastewartia (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; Optionally, it is E. coli.

9. A method for producing D-mannitol, comprising using the engineered cell according to any one of the preceding claims to ferment and produce D-mannitol; Optionally, the method further comprises synthesizing D-mannitol using glucose as a substrate.

10. Use of an engineered cell according to any preceding claim in the production of D-mannitol.

Images