A method for synthesizing fucose using multiple carbon sources and its application

Abstract

This invention provides an *E. coli* strain capable of synthesizing L-fucose using various raw materials. The *E. coli* strain underwent multiple genetic engineering modifications, including knocking out genes such as fucP, enhancing genes such as manA, and introducing genes such as upp. This *E. coli* strain can synthesize L-fucose at a high level using sucrose, glycerol, glucose, and other raw materials, and has promising industrial applications in biomanufacturing and food ingredient production.

Description

Technical Field

[0001] This invention relates to the construction and application of a recombinant *Escherichia coli* strain for the synthesis of L-fucose, belonging to the field of bioengineering technology. The engineered strain uses *Escherichia coli* as the starting strain and integrates a product degradation module, a precursor synthesis module, a cofactor supply module, and a target compound generation module to provide a method and application for synthesizing L-fucose using multiple carbon sources. Background Technology

[0002] Fucose (also known as 6-deoxy-L-galactose), a hexacarbon sugar, is a naturally occurring methylpentose existing in its L-isomer form. It is found in oligosaccharides, polysaccharides, and glycosides in bacteria and plants, and is an important component of mammalian cytosaccharides and glycolipids. It is one of the five basic monosaccharides that make up human milk oligosaccharides (HMOs). Furthermore, fucose monomers can polymerize to form fucoidan, which is mainly found in brown algae. In medicine, it has anti-tumor effects and can alleviate intestinal diseases; in the food industry, it has the potential to be used as an emulsifier and is widely used in food and infant formula. Numerous studies have evaluated its safety, and experimental results indicate that L-fucose can be used as a food-grade functional ingredient.

[0003] Fucose can be chemically synthesized from inexpensive monosaccharides, but chemical synthesis is time-consuming, environmentally unfriendly, and yields low output. L-fucose can be efficiently synthesized from fucoidan using acid hydrolysis and efficient separation techniques. L-fucose can also be obtained through enzymatic reactions using L-fucose as a substrate, but the substrate L-fucose is more expensive than the product L-fucose, thus limiting its application to industrial production. Currently, microbial synthesis of L-fucose based on metabolic engineering techniques and strategies is highly efficient and environmentally friendly. The synthesis of 2'-fucoyllactose using exogenously added lactose as a glycosyl acceptor, and the hydrolysis of 2'-fucoyllactose into L-fucose and lactose by introducing specific α-L-fucosidase, are widely used. However, this method requires the introduction of additional lactose as a glycosyl acceptor in addition to carbon sources such as glycerol or glucose. Therefore, designing efficient catalytic reaction systems using inexpensive substrates such as glucose and sucrose as carbon sources holds promise for significantly reducing production costs. However, the core challenge of this strategy lies in the construction of highly efficient microbial strains, which remains a major technical bottleneck. Especially in the de novo synthesis of L-fucose, factors such as raw material selection, metabolic pathway efficiency, and host strain performance collectively constrain its synthesis efficiency, making it difficult to achieve high-level mass production. Summary of the Invention

[0004] To address the existing technical difficulties and problems, this invention provides a method for constructing and applying a recombinant Escherichia coli strain for synthesizing L-fucose.

[0005] A first aspect of the present invention provides a recombinant Escherichia coli strain for synthesizing L-fucose, said recombinant Escherichia coli strain comprising at least one or more of the following modifications:

[0006] (1) Introduce the nucleotide sugar diphosphatase UPP gene;

[0007] (2) Introduce the dephosphatase PPase gene;

[0008] (3) Enhance the expression of the glucose-6 phosphate dehydrogenase gene zwf;

[0009] (4) Enhance the expression of the guanylate kinase gene gmk;

[0010] (5) Enhance the expression of the nucleoside diphosphate kinase gene ndk;

[0011] Furthermore, the recombinant Escherichia coli strain also includes at least one or more of the following modifications:

[0012] (6) Enhance the expression of the mannose-6 phosphate isomerase gene manA;

[0013] (7) Enhance the expression of the guanylate kinase gene cpsG;

[0014] (8) Enhance the expression of the guanylate kinase gene cpsB;

[0015] (9) Enhance the expression of the guanylate kinase gene gmd;

[0016] (10) Enhance the expression of the guanylate kinase gene fcI;

[0017] Furthermore, the recombinant Escherichia coli strain also includes at least one or more of the following modifications:

[0018] (11) Knock out the L-fucose transporter gene fucP;

[0019] (12) Knock out the L-fucose isomerase gene fucI;

[0020] (13) Knock out the L-fucokinase gene fucK;

[0021] (14) Knockout of the undecylylphosphoglucose-1-phosphotransferase gene wcaJ;

[0022] (15) Knock out the 6-phosphofructokinase 1 gene pfkA;

[0023] (16) Knock out the 6-phosphofructokinase 2 gene pfkB;

[0024] (17) Knock out the clavatin biosynthesis acetyltransferase gene wcaF;

[0025] (18) Knock out the GMP reductase gene guaC.

[0026] In one implementation, the above-mentioned modifications to enhance (3), (4), (5), (6), (7), (8), (9), and (10) can be specifically achieved by regulating the expression of related genes on chromosomes using constitutive strong promoters.

[0027] In one embodiment, the constitutive strong promoter may further be the PJ23119 promoter.

[0028] In one embodiment, the modifications described in (11), (12), and (13) above can be to knock out the entire fucPIK gene cluster on the chromosome.

[0029] In one implementation, the modifications in (6) and (15) above can specifically be to integrate P while knocking out pfkA. J23119 The promoter initiates the expression of the fructose-6-phosphate isomerase-encoding gene manA.

[0030] In one implementation, the modifications described in (7), (8), and (16) above can specifically be to integrate P while knocking out pfkB. J23119 The promoter initiates the expression of the gene clusters cpsG-cpsB.

[0031] In one implementation, the modifications described in (9), (10), and (17) above can specifically involve integrating P while knocking out wcaF. J23119 The promoter initiates the expression of the gene cluster gmd-fcI.

[0032] In one implementation, the modifications described in (4), (5), and (18) above can specifically be to integrate P while knocking out guaC. J23119 The promoter initiates the expression of the gene cluster gmk-ndk.

[0033] In one embodiment, the modifications in (1) and (2) above can be specifically achieved by overexpressing nucleotide sugar diphosphatase UPP and dephosphatase PPase using the pBAD / HisB plasmid.

[0034] In one embodiment, the nucleotide sequence of the nucleotide sugar diphosphatase UPP is shown in SEQ ID NO. 34, 36. The nucleotide sequence of the dephosphatase PPase is shown in SEQ ID NO. 38, 40, 42, 44.

[0035] According to a second aspect of the present invention, a method for producing L-fucose using different carbon sources, such as sucrose, glucose, glycerol, and fructose substrates, is provided, the method being to produce L-fucose by fermentation using the recombinant Escherichia coli.

[0036] In one embodiment, the recombinant *E. coli* is reacted to produce L-fucose according to the following steps: 1) L-fucose shake-flask fermentation process: The constructed strain is inoculated into LB liquid medium and cultured overnight at 37 ℃ and 200 rpm to obtain seed culture. The seed culture is then inoculated into 100 mL of fresh medium at a ratio of 1:100 and cultured at 37 ℃ and 200 rpm until the OD600 reaches 0.8–1.0. IPTG is added to a final concentration of 0.2 mM, and induction is carried out overnight for 16 h. 2) The overnight induced bacterial culture is centrifuged at 5000 rpm for 10 min to collect the bacterial cells. Simultaneously, substrates (sucrose, glucose, glycerol, fructose) and 2 mM Mg2+ are added to the reaction system to a final concentration of 20 g / L. 2+ 1 X M9 reacted in a shake flask for 6 h to produce L-fucose.

[0037] In one embodiment, the 5 x M9 salt solution of the reaction system is formulated as follows: 12.8 g Na2PO4·7H2O, 3.0 g KH2PO4, 0.5 g NaCl, and 1.0 g NH4Cl, dissolved in 200 ml double-distilled water, and sterilized at 121 degrees Celsius for 15 minutes.

[0038] A third object of the present invention is to provide the use of the said Escherichia coli strain in the preparation of L-fucose or products containing L-fucose.

[0039] Compared with the prior art, the method for synthesizing L-fucose and the construction and application of recombinant Escherichia coli provided by the present invention have the following advantages:

[0040] 1) The technical solution of this invention constructs a recombinant Escherichia coli and achieves the de novo synthesis of L-fucose for the first time using different carbon sources as direct substrates without adding other exogenous substances;

[0041] 2) The technical solution of this invention constructs and ferments recombinant Escherichia coli to produce L-fucose using a relatively inexpensive carbon source, which is more green and environmentally friendly than the traditional acid hydrolysis method for producing L-fucose.

[0042] In summary, this invention provides a method for synthesizing L-fucose and the construction and application of recombinant Escherichia coli, which has the ability to synthesize the target compound in a short time and with high efficiency, providing a foundation for large-scale industrial production. Attached Figure Description

[0043] Figure 1 The liquid chromatography spectrum of L-fucose standard;

[0044] Figure 2 Liquid chromatography chromatograms of standards for substrates glucose, fructose, sucrose, and glycerol. Detailed Implementation

[0045] definition

[0046] Unless otherwise defined 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.

[0047] Throughout this specification and the appended claims, the terms "comprising" and "including," as well as variations thereof, shall be interpreted inclusively. That is, where the context permits, these terms are intended to express the possibility of including other elements or integers not specifically listed.

[0048] The article “a / an” is used in this text to refer to one / an or more than one / an (i.e., one / an 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 / an element. When a noun is mentioned in the singular (e.g., compound, additive, etc.), the plural form is intended to be included. Therefore, when referring to a specific part (e.g., “gene”), unless otherwise specified, it means “at least one” of the genes, e.g., “at least one gene”.

[0049] Unless otherwise expressly indicated, the various embodiments of the invention described herein can be combined in various ways.

[0050] The term "carbon source" refers to a source of carbon, preferably a compound or molecule containing carbon. Preferably, the carbon source is a carbohydrate, amino acid, or its derivative. In this document, a carbon source is understood as an organic compound composed of elements such as carbon, oxygen, and hydrogen.

[0051] The term "cell" refers to a eukaryotic or prokaryotic organism, preferably existing as a single cell. In this invention, the cell can be recombinant *Escherichia coli*. That is, the recombinant cell is selected from a cell population of a genus of *Escherichia coli*.

[0052] As used herein, the term “recombinant” / “engineered” (e.g., references to “recombinant E. coli,” “recombinant cell,” “recombinant microorganism,” and / or “recombinant strain”) can refer to a cell, microorganism, or strain containing nucleic acids as a result of one or more gene modifications. Simply put, a cell, microorganism, or strain contains different combinations of nucleic acids from one or more of its parents (any one of them). To construct recombinant cells, microorganisms, or strains, one or more recombinant DNA techniques and / or another one or more mutagenesis techniques can be used. For example, recombinant E. coli and / or recombinant E. coli cells may contain nucleic acids not present in the corresponding wild-type E. coli and / or cells, which have been introduced into the E. coli or E. coli cells using recombinant DNA techniques, or the absence of the nucleic acid in the wild-type E. coli and / or cells is the result of one or more mutations 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 (e.g., using recombinant DNA techniques or another mutagenesis technique such as UV irradiation). Furthermore, the term “recombinant” can suitably refer to, for example, cells, microorganisms, or strains from which nucleic acid sequences have been removed using recombinant DNA techniques.

[0053] In this paper, "recombinant E. coli containing or having a certain activity" is understood to mean that recombinant E. coli can contain one or more nucleic acid sequences encoding proteins having such activity. Therefore, recombinant E. coli is permitted to functionally express such proteins or enzymes.

[0054] The term "functional expression" refers to functional transcription in which the relevant nucleic acid sequence is present, allowing the nucleic acid sequence to actually be transcribed, for example, leading to protein synthesis.

[0055] As used herein with respect to proteins or peptides, the term "mutation" means that, compared to the wild-type or naturally occurring protein or peptide sequence, at least one amino acid has been substituted, inserted into, or deleted from the amino acid sequence. Amino acid substitutions, insertions, or deletions can be achieved, for example, by mutagenesis of the nucleic acids encoding those amino acids. Mutagenesis is a method well known in the art and includes, for example, site-directed mutagenesis via PCR or via oligonucleotide-mediated mutagenesis, as described in: Sambrook et al., Molecular Cloning—A Laboratory Manual, 2nd ed., Volumes 1–3 (1989), published by Cold Spring Harbor Publishing.

[0056] As used herein with respect to genes, the term "mutation" means that, compared to a wild-type or naturally occurring nucleic acid sequence, at least one nucleotide in the nucleic acid sequence of a gene or its regulatory sequence has been replaced, inserted into, or deleted from the nucleic acid sequence by a different nucleotide. The substitution, insertion, or deletion of amino acids can be achieved, for example, via mutagenesis, resulting in transcription of a protein sequence having a qualitatively or quantitatively altered function, or the knockout of the gene. In the context of this invention, "altered gene" has the same meaning as a mutated gene.

[0057] As used herein, the term "gene" refers to a nucleic acid sequence of mRNA that can be transcribed and then translated into a protein. A gene that encodes a protein is one or more nucleic acid sequences that encode that protein.

[0058] As used herein, the term "nucleic acid" or "nucleotide" refers to a monomeric unit in a single-stranded or double-stranded deoxyribonucleotide or ribonucleotide polymer (i.e., a polynucleotide), and unless otherwise limited, encompasses known analogs that have the inherent properties of natural nucleotides because they hybridize with single-stranded nucleic acids (e.g., peptide nucleic acids) in a manner similar to naturally occurring nucleotides. For example, an enzyme defined by the nucleotide sequence encoding an enzyme includes (unless otherwise limited) a nucleotide sequence that hybridizes with a reference nucleotide sequence encoding the enzyme. A polynucleotide can be a natural or heterologous structure or a full-length or subsequence of a regulatory gene. Unless otherwise indicated, the term includes references to the specified sequence and its complementary sequence. Thus, DNA or RNA having a backbone modified for stability or other reasons is a "polynucleotide" as contemplated herein. Furthermore, DNA or RNA containing rare bases (such as inosine) or modified bases (such as triphenylmethylated bases) (to name just two examples) is a polynucleotide as used herein. It will be understood that DNA and RNA have been modified in a wide variety of ways for many useful purposes known to those skilled in the art. As used herein, the term polynucleotide includes such chemical, enzymatic, or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA specific to viruses and cells, including both simple and complex cells.

[0059] The terms "nucleotide sequence" and "nucleic acid sequence" are used interchangeably in this article. An example of a nucleic acid sequence is a DNA sequence.

[0060] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers, for example, those containing amino acid residues as shown in the amino acid sequence. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. An essential property of such analogs of naturally occurring amino acids is that, when incorporated into a protein, the protein exhibits a specific reactivity to antibodies induced by proteins composed entirely of the same, but entirely, naturally occurring amino acids. The terms “polypeptide,” “peptide,” and “protein” also include modifications, including but not limited to glycosylation, lipid attachment, sulfation, γ-carboxylation, hydroxylation, and ADP-ribosylation of glutamate residues.

[0061] The term "enzyme" in this document refers to a protein that has a catalytic function. In cases where a protein catalyzes a biological reaction, the terms "protein" and "enzyme" may be used interchangeably herein. When referring to enzymes in the Enzyme Classification (EC), an enzyme class is a category in which an enzyme is classified or can be classified according to the enzyme nomenclature provided by 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 are not yet (not yet) classified in the specified category but can be so.

[0062] If a protein or nucleic acid sequence (such as a gene) is referred to in this document by reference accession number, unless otherwise specified, that number is specifically used to refer to a protein or nucleic acid sequence (gene) that can be found via www.ncbi.nlm.nih.gov / (available as of October 1, 2020).

[0063] Each nucleic acid sequence encoding a polypeptide in this paper also includes variants of any conserved modifications thereof. This includes, by reference to the genetic code, every possible silent variant of the nucleic acid. The term “conserved variant” applies to both amino acids and nucleic acid sequences. With respect to a particular nucleic acid sequence, a conserved variant refers to those nucleic acids that encode the same amino acid sequence or a variant of a conserved modified amino acid sequence 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, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Therefore, at each position where the codon specifies alanine, the codon can be changed to any of the described corresponding codons without changing the encoded polypeptide. Such nucleic acid variations are “silent variants” and represent a variant of a conserved modification.

[0064] As used herein, the term "functional homolog" (or "homolog") of a gene having a specific sequence (e.g., "SEQ ID NO:X") refers to a polypeptide and / or amino acid sequence containing the specific sequence, or a nucleic acid sequence containing 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 the (qualitative) same enzymatic function for substrate transformation.

[0065] As used herein, the term "functional homolog" (or simply "homolog") 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 containing said specific sequence, provided that one or more nucleic acids are mutated, substituted, deleted, added, and / or inserted, and that the polynucleotide encodes a polypeptide sequence having the (qualitative) same enzymatic function for substrate transformation. With respect to nucleic acid sequences, the term "functional homolog" 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.

[0066] In this document, sequence identity is defined as the relationship between two or more amino acid (peptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by sequence comparison. Typically, sequence identity or similarity is compared over the entire length of the sequences being compared. In this art, “identity” also means the degree of sequence correlation between amino acid or nucleic acid sequences, as determined by matching strings of such sequences.

[0067] Amino acid or nucleotide sequences are said to be homologous when they exhibit a certain level of similarity. Two sequences being homologous indicates a common evolutionary origin. Whether two homologous sequences are closely related or more distantly related is indicated by "identity percentage" or "similarity percentage," which are high or low, respectively. Although controversial, "homology level" or "homology percentage" is often used interchangeably to indicate "identity percentage" or "similarity percentage." Sequence comparisons and the determination of the identity percentage between two sequences can be accomplished using mathematical algorithms. Technicians will become aware of the fact that several different computer programs can be used to align two sequences and determine the homology between them (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 J.B. Kruskal, "Time warps, string edits and macro molecules: the theory and practice of sequence comparison", (1983), pp. 1-44, published by Addison-Wesley Publishing Company, Massachusetts USA).

[0068] The Needleman and Wunsch algorithm can be used to align two sequences to determine the percentage of identity between the two amino acid sequences. (Needleman et al., "A General Method Applicable to the Search for Similarities in the Amino Acid Sequence of Two Proteins" (1970), J. Mol. Biol., Vol. 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 this invention, the NEEDLE program from the EMBOSS package (version 2.8.0 or later, 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 / ) was used. For protein sequences, EBLOSUM62 was used as the substitution matrix. For nucleotide sequences, EDNAFULL was used. Other matrices may be specified. Optional parameters for amino acid sequence alignment are a vacancy opening penalty of 10 and a vacancy expansion penalty of 0.5. Those skilled in the art will understand that all these different parameters will produce slightly different results, but the overall percentage of identity between the two sequences does not change significantly when different algorithms are used.

[0069] Homology or identity is the percentage of identical matches between two complete sequences across the total aligned region, including any vacancies or extensions. Homology or identity between two aligned sequences is calculated as follows: the number of corresponding positions in both sequences showing the same amino acid in the alignment divided by the total length of the alignment, including vacancies. Identity, as defined herein, is available from NEEDLE and is labeled "IDENTITY" in the program's output.

[0070] Homology or identity between two aligned sequences is calculated as follows: the number of corresponding positions in both sequences showing the same amino acid in the alignment is divided by the total length of the alignment after subtracting the total number of vacancies in the alignment. Identity as defined herein can be obtained from NEEDLE using the NOBRIEF option and is marked as "longest-identity" in the program's output.

[0071] 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 (e.g., in the sequence listing).

[0072] Optionally, when determining the degree of amino acid similarity, those skilled in the art may also consider so-called “conservative” amino acid substitutions, which will be clear to them. Conservative amino acid substitution refers to the interchangeability of residues with similar side chains. For example, a group of amino acids with aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids with aliphatic-hydroxy side chains is serine and threonine; a group of amino acids with amide-containing side chains is asparagine and glutamine; a group of amino acids with aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids with basic side chains is lysine, arginine, and histidine; and a group of amino acids with sulfur-containing side chains is cysteine ​​and methionine. In one embodiment, the group of conserved amino acid substitutions is: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. 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 has been inserted at its position. Preferably, the amino acid changes are conserved. In one embodiment, the conserved substitutions for 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.

[0073] The nucleotide sequences of the present invention can also be defined by their ability to partially hybridize with 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 nucleic acid sequences of at least about 25 nucleotides, preferably about 50, 75, or 100 nucleotides, and most preferably about 200 or more nucleotides, to hybridize at about 65°C in a solution containing about 1 M salt (preferably 6x SSC or any other solution with equivalent ionic strength), and to be washed at 65°C in a solution containing about 0.1 M or less salt (preferably 0.2x SSC or any other solution with equivalent 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 about 90% or higher sequence identity. In this document, moderate conditions are defined as conditions that allow nucleic acid sequences of at least 50 nucleotides, preferably about 200 or more nucleotides, to hybridize at about 45°C in a solution containing about 1M salt (preferably 6x SSC or any other solution with equivalent ionic strength), and to wash at room temperature in a solution containing about 1M salt (preferably 6x SSC or any other solution with equivalent 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 to specifically identify sequences with identity varying between 50% and 90%.

[0074] "Expression" refers to the transcription of genes into structural RNA (rRNA, tRNA) or messenger RNA (mRNA), which is then translated into proteins.

[0075] "Overexpression" refers to the expression of a gene (corresponding nucleic acid sequence) in recombinant cells exceeding its expression in the corresponding wild-type cells. Such overexpression can be arranged, for example, by increasing the transcription frequency of one or more nucleic acid sequences, such as by operatively linking the nucleic acid sequence to a functional promoter in the recombinant cell; and / or by increasing the copy number of a nucleic acid sequence.

[0076] The term "upregulation" and its variations refer to the process by which cells increase the amount of cellular components, such as RNA or proteins. This upregulation can be in response to or caused by gene modification.

[0077] In this article, the term "pathway" or "metabolic pathway" is understood as a series of chemical reactions that build up and break down molecules in the cell.

[0078] Nucleic acid sequences (i.e., polynucleotides) or proteins (i.e., polypeptides) can be native or heterologous to the host cell's genome.

[0079] The terms "natural," "homologous," or "endogenous" in relation to a host cell mean that the nucleic acid sequence is indeed naturally present in the host cell's genome, or that the protein is naturally produced by that cell. The terms "natural," "homologous," and "endogenous" are used interchangeably in this document.

[0080] As used herein, “heterologous” or “exogenous” can refer to a nucleic acid sequence or a protein. For example, in relation to a host cell, “heterologous” can 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 a foreign species, or, if from the same species, substantially modified relative to its natural form in terms of composition and / or genomic loci through deliberate human intervention. For example, a promoter operatively linked to a natural structural gene is derived 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 can be derived from a foreign species, or, if from the same species, substantially modified relative to its original form through deliberate human intervention. That is, heterologous protein expression involves the expression of a protein that is not naturally expressed in the host cell in this manner. The term “heterologous expression” refers to the expression of a heterologous nucleic acid 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 proteins or enzymes with specific activities can be expressed in such eukaryotic systems. In some embodiments, transformed / transfected cells can be used as expression systems for enzymes. The expression of heterologous proteins in *E. coli* is well known. *The Cold Spring Harbor Laboratory* is a recognized work describing various methods that can be used to express proteins in *E. coli*.

[0081] As used herein, a "promoter" is a DNA sequence that directs the transcription of a (structural) gene or other (partial) nucleic acid sequence. Appropriately, a promoter is located in the 5' region of a gene, near the transcription start site of the (structural) gene. Promoter sequences can be constitutive, inducible, or repressive. In one embodiment, no (external) inducer is required.

[0082] As used herein, the term "vector" includes references to autosomal expression vectors and integration vectors for integration into chromosomes.

[0083] The term "expression vector" refers to a linear or circular DNA molecule containing a segment encoding a target polypeptide, which is controlled (i.e., operatively linked to) additional nucleic acid segments that provide for its transcription. These additional segments may include promoter and terminator sequences and may optionally include one or more origins of replication, one or more selection markers, enhancers, polyadenylation signals, etc. Expression vectors are typically derived from plasmid or viral DNA, or may contain elements of both.

[0084] "Plasmid" refers to autonomously replicating extrachromosomal DNA that does not integrate into the genome of a microorganism and is usually circular in nature.

[0085] In this paper, "host cell" is understood to be a cell (such as an E. coli cell) that will be transformed with one or more nucleic acid sequences encoding one or more heterologous proteins to construct a transformed cell (also known as a recombinant cell). For example, a transformed cell may contain a vector and may support the replication and / or expression of the vector.

[0086] As used herein, “transformation” refers to the insertion of a foreign polynucleotide into a host cell, regardless of the method used for insertion, such as direct uptake, transduction, f-conjugation, or electroporation. The foreign polynucleotide may be maintained as a non-integrating vector (e.g., a plasmid) or alternatively integrated into the host cell genome.

[0087] The present disclosure will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present disclosure and are not intended to limit the scope of the present disclosure. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following embodiments are commercially available.

[0088] Those skilled in the art will understand that the gene or its functional homologs disclosed herein comprise a nucleic acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the exemplarily listed sequences, or having one or more mutated, substituted, inserted, and / or deleted amino acid sequences compared to the amino acid sequence it encodes.

[0089] Preferably, compared with the amino acid sequence encoded by a gene, the amino acid sequence encoded by any functional homolog of that 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.

[0090] Exemplary, and not limiting, the functional homolog of the BJUPP gene shown in SEQ ID NO. 34 comprises a nucleic acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any of those nucleic acid sequences, or its functional homolog comprises a nucleic acid sequence having one or more mutations, substitutions, insertions, and / or deletions compared to any of those nucleic acid sequences; preferably, compared to such nucleic acid sequences, the nucleic acid sequence of any such functional homolog has no more than 300, 250, 200, 150, 100, 75, 50, 40, 30, 20, 10, or 5 nucleic acid mutations, substitutions, insertions, and / or deletions. More preferably, the functional homolog of the BJUPP gene 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. 35.

[0091] Those skilled in the art will understand that other sequences include the above-described cases.

[0092] Unless otherwise specified herein, the terms and terminology used herein should be understood in accordance with the conventional knowledge and usage of those skilled in the art. Unless otherwise stated, the specific operational methods (including: preparation processes, experimental steps, detection methods, etc.) employed in this application utilize conventional techniques in the fields of biochemistry, cell biology, molecular biology, gene editing (e.g., recombinant DNA technology), zoology, and related areas. These techniques are well-described in existing literature. For details, please refer to 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 Press, updated irregularly; Kursad Turksen et al., *Embryonic StemCell 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.

[0093] The Trans1-T1 competent cells used in the following examples are products of Beijing TransGen Biotech, with product catalog number CD501.

[0094] Carrier pBAD / HisB: Invitrogen, catalog number V430-01.

[0095] L-Fucose: Purchased from Aladdin, catalog number F110930

[0096] Sugar: Purchased from Aladdin, catalog number S112224

[0097] Fructose: Purchased from Aladdin, catalog number F108332

[0098] Glucose: Purchased from Aladdin, catalog number G116302

[0099] Glycerin: Purchased from Aladdin, catalog number G116208 Detailed Implementation

[0100] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0101] The plasmids, restriction enzymes, PCR enzymes, column-based DNA extraction kits, DNA gel recovery kits, Gibson ligation kits, and other commercially available products used in the following examples were performed according to the kit instructions. Conventional procedures such as colony PCR, nucleic acid agarose gel electrophoresis, heat shock transformation, electroporation, preparation of competent cells, and extraction and preservation of bacterial genomes were performed according to Molecular Cloning: A Laboratory Manual. Sequencing of the plasmids and DNA products was performed by Qingke Biotechnology (Tianjin). The vector pBAD / HisB used in the following examples was a product of Invitrogen, catalog number V430-01; Escherichia coli DH5α was purchased from Beijing Qingke Biotechnology Co., Ltd., catalog number TSC01.

[0102] 1. The specific culture medium and reaction system components used in this invention are as follows:

[0103] (1) LB liquid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride.

[0104] (2) LB solid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar powder.

[0105] (3) Reaction system: 20 g / L glucose, 20 g / L fructose, 20 g / L sucrose or 20 g / L glycerol, 1 × M9 salt solution, 2 mM Mg 2+ .

[0106] (4) Antibiotic concentrations: streptomycin 50 mg / L (liquid culture medium), streptomycin 50 mg / L (solid culture medium), kanamycin 50 mg / L, ampicillin 100 mg / L.

[0107] 2. The fermentation process of L-fucose in this invention is as follows:

[0108] (1) L-fucose shake-flask fermentation process: The constructed strain was inoculated into LB liquid medium and cultured overnight at 37℃ and 200 rpm to obtain seed culture. The seed culture was then inoculated into 100 mL of fresh medium at a ratio of 1:100 and cultured at 37℃ and 200 rpm until OD. 600 The concentration was 0.8–1.0, and IPTG was added to a final concentration of 0.2 mM. Induction was performed overnight for 16 h.

[0109] (2) Collect the bacterial cells by centrifuging the overnight induced bacterial culture at 5000 rpm for 10 min, and simultaneously add substrate to a final concentration of 20 g / L and 2 mM Mg. 2+ 1 X M9 reacted in a shake flask for 6 h to produce L-fucose.

[0110] 3. The method for detecting L-fucose in this invention is as follows:

[0111] Take 1 mL of fermentation broth, centrifuge at 12000 rpm for 10 min, collect the supernatant, and use it for HPLC determination.

[0112] HPLC detection conditions: Shimadzu high performance liquid chromatography (HPLC) system; column: 87 C column; detector: RID differential detector; mobile phase: H2O; flow rate: 0.6 mL / min; column temperature: 60℃; injection volume: 10 μL.

[0113] The liquid chromatography peak spectrum of the L-fucose standard is as follows: Figure 1 As shown, the liquid chromatography peak spectra of the substrates glucose, fructose, sucrose, and glycerol standards are as follows: Figure 2 As shown.

[0114] 4. The plasmids and strains described in this invention are as follows:

[0115] The construction and editing of chassis strains were carried out using the dual plasmid gene editing system pCas / pTargetF, which has been published in the literature: Acta Biochim BiophysSin, 2021, 53(5), 620-627.

[0116] Expression vector plasmids were constructed using the Gibson assembly kit.

[0117] Table 1. Strains and plasmids involved in the following examples.

[0118] MG1655 The Genetic Collection Center for E. coli at Yale University (CGSC#: 6300) BW25113 The Genetic Collection Center for E. coli at Yale University (CGSC#: 7636) MC02 China General Microbiological Culture Collection Center (CGMCC) (accession number CGMCC No. 34378) FUC000 MC02∆fucI∆fucP∆fucK∆wcaJ FUC001 FUC000ΔpfkA::PJ23119-manAΔpfkB::PJ23119- cpsG-cpsB ΔwcaF::PJ23119-CpsG-CpsB-gmd-fcI FUC002 <![CDATA[FUC001∆P zwf ::P J23119 ∆guaC::P J23119 -ndk-gmk-TrrB]]> L01 FUC002 / pBAD / HisB-BJUPP-yiHX L02 FUC002 / pBAD / HisB-BJUPP-yieH L03 FUC002 / pBAD / HisB-BJUPP-hxpA L04 FUC002 / pBAD / HisB-BJUPP-hxpB L05 FUC002 / pBAD / HisB-AMUPP-yiHX L06 FUC002 / pBAD / HisB-AMUPP-yieH L07 FUC002 / pBAD / HisB-AMUPP-hxpA L08 FUC002 / pBAD / HisB-AMUPP-hxpB

[0119] 5. The primers used in this invention are as follows:

[0120] Table 2 shows the primers required in the following examples.

[0121] fucP-up-F ttcgttccattagctacctctctctga fucK-down-R ccccatcattcgataaccgcccgcgtcatg wcaJ-up-F cgcaggtggcacgccggtaatg wcaJ-down-R cagcggccagaaatgggcgctaaccaccgt pfkA-up-F cgttgggtggtgcggcggacg pfkA-down-R ctttatcaatccgcccgcgttccg pfkB-up-F ttcagagttccatgtgacgttgtaag pfkB -down-R atccgttgttcagcaaaaaacgttgaccag wcaF-up-F cgtctgtccgtttattttgcgaaaagc wcaF-down-R cacgcagggaatccatattgccgagg zwf-up-F taaaatcaggaaaaccgcgcgtgtccatg zwf-down-R ccgtttctttaccaagatagtggtcgat guaC-up-F cgatgcgatagcatccggtgcg guaC-down-R ctaccggcactaacgcaggggat N20 primers The underlined part represents the N20 sequence. fucI-N20-F <![CDATA[actagt ctgctgcgctttgcccgcgc gttttatagctagaaatagcaagttaaaataag]]> fucI-N20-R <![CDATA[ctctaaaac gcgcgggcaaagcgcagcag actagtattatacctaggactgagctagc]]> wcaJ-N20-F <![CDATA[actagt gcgcatggtcgcggtggcgg gttttatagctagaaatagcaagttaaaataag]]> wcaJ-N20-R <![CDATA[ctctaaaac ccgccaccgcgaccatgcgc actagtattatacctaggactgagctagc]]> pfkA-N20-F <![CDATA[actagt gaagtaatgggtatttatga gttttatagctagaaatagcaagttaaaataag]]> pfkA-N20-R <![CDATA[gctctaaaac tcataaatacccattacttc actagtattatacctaggactgagctagc]]> pfkB-N20-F actagtactgatttccgctgcgcaaagttttagagctagaaatagcaagttaaaataag pfkB-N20-R <![CDATA[gctctaaaac tttgcgcagcggaaatcagt actagtattatacctaggactgagctagc<!-- 11 --> ]]> wcaF-N20-F <![CDATA[actagt gcttttttattacgtttatt gttttatagctagaaatagcaagttaaaataag]]> wcaF-N20-R <![CDATA[gctctaaaac aataaacgtaataaaaaag cactagtattatacctaggactgagctagc]]> zwf-N20-F <![CDATA[actagt ccactatcttggtaaagaaa gttttatagctagaaatagcaagttaaaataag]]> zwf-N20-R <![CDATA[gctctaaaac tttctttaccaagatagtgg actagtattatacctaggactgagctagc]]> guaC-N20-F <![CDATA[actagt acagattctcgacctgaacc gttttatagctagaaatagcaagttaaaataag]]> guaC-N20-R <![CDATA[gctctaaaac ggttcaggtcgagaatctgt actagtattatacctaggactgagctagc]]> Primers for plasmid construction pYB1S-F gaattcggtgagctcggtctgcagc pYB1S-R ctcgaggctgccgcgcggcaccaggc pYB1S-BJUPP-F tggtgccgcgcggcagcctcgagatgtctgatcgctccggcccaaacc BJUPP-R catggtatatctccttttaggtagaatggttagaggagctagcg BJUPP-yiHX-F ttctacctaaaaggagatataccatgctctatatctttgatttag pYB1S-yiHX-R agctgcagaccgagctcaccgaattcttagcataacaccttcgcgaaatagtcc BJUPP-yieH-F ttctacctaaaaggagatataccatgtcccggatagaagcggtatttttc pYB1S-yieH-R agctgcagaccgagctcaccgaattcctatgccgtaatatcccaaccacgc BJUPP-hxpA-F taaaaggagatataccatgcggtgcaaaggttttctgtttg pYB1S-hxpA-R agctgcagaccgagctcaccgaattctcactgaataataacatcgccatttgg BJUPP-hxpB-F ttctacctaaaaggagatataccatgtcaaccccgcgtcagattc pYB1S-hxpB-R tgcagaccgagctcaccgaattcttaaccgagaaggtcttttgcgg pYB1S-AMUPP-F gcgcggcagcctcgagatggctactacctggggcgctgc AMUPP-R catggtatatctccttttagtgagagatacccagggactg AMUPP-yiHX-F tctctcactaaaaggagatataccatgctctatatctttgatttaggtaatgtg pYB1S-yiHX-R agctgcagaccgagctcaccgaattcttagcataacaccttcgcgaaatagtcc AMUPP-yieH-F tctctcactaaaaggagatataccatgtcccggatagaagcggtatttttc pYB1S-yieH-R agctgcagaccgagctcaccgaattcctatgccgtaatatcccaaccacgc AMUPP-hxpA-F tctctcactaaaaggagatataccatgcggtgcaaaggttttctgtttg pYB1S-hxpA-R agctgcagaccgagctcaccgaattctcactgaataataacatcgccatttgg AMUPP-hxpB-F tctctcactaaaaggagatataccatgtcaaccccgcgtcagattcttg pYB1S-hxpB-R tgcagaccgagctcaccgaattcttaaccgagaaggtcttttgcgg pYB1S-VF aagattagcggatcctacctg pYB1S-VR tggcagttccctactctc

[0122] Table 3 Sequence of the present invention

[0123] SEQ ID NO.2 fucI L-fucose isomerase Gene ID:946195 / NP_417282.1 SEQ ID NO.3 fucK L-fucose kinase Gene ID:946022 / NP_417283.2 SEQ ID NO.4 wcaJ Undecaprenyl-phosphate-glucose-1-phosphate transferase Gene ID:946583 / NP_416551.1 SEQ ID NO.5 pfkA 6-Phosphokinase 1 Gene ID: 948412 / NP_418351.1 SEQ ID NO.6 pfkB 6-Phosphofructokinase 2 Gene ID: 9462306 / NP_416237.3 SEQ ID NO.7 manA Fructose-6-phosphate isomerase Gene ID: 944840 / NP_416130.3 SEQ ID NO.8 wcaF Claritase biosynthesis acetyltransferase Gene ID: 946578 / NP_416558.1 SEQ ID NO.9 cpsG Phosphomannose mutase Gene ID: 946574 / NP_416552.1 SEQ ID NO.10 cpsB mannose-1-phosphate guanine transferase Gene ID: 946580 / NP_416553.1 SEQ ID NO.11 gmd GDP-mannose 4,6-hydrolase Gene ID: 946562 / NP_416557.1 SEQ ID NO.12 fcI GDP-L-fucose synthase Gene ID: 946563 / NP_416556.1 SEQ ID NO.13 zwf NADP+-dependent glucose-6-phosphate dehydrogenase Gene ID: 946370 / NP_416366.1 SEQ ID NO.14 guaC GMP reductase Gene ID is 948986 / NP_414646.1 SEQ ID NO.15 gmk Guanosine kinase Gene ID: 948163 / NP_418105.1 SEQ ID NO.16 ndk Nucleoside diphosphate kinase Gene ID: 945611 / NP_417013.1 SEQ ID NO.17 P119 atggcttgtcatgcttaattgacagctagctcagtcctaggtataatgctagcaggggagaccacaacggtttccctctacaaataattttgtttaactttcgcgcgcgtaacaggaggaattaacc SEQ ID NO.18 RBS aggaggaattaacc SEQ ID NO.19 TrrnB tgcctggcggcagtagcgcggtggtcccacctgaccccatgccgaactcagaagtgaaacgccgtagcgccgatggtagtgtggggtctccccatgcgagagtagggaactgccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctt SEQ ID NO.20 fucPup ttcgttccattagctacctctctctgattcaaaacagggcaataatgttgttcctttcacactattgaattagccgtttaattacccaccatcttcttcctgattaacaagaaagaaattcacaagcttatattttgtgacctggttcaactaatcacagtaaataactgcaagttctctttttataaccccattaaaaatgaccgctcttaaaaatatttatcaaaacggtcatttttctattcctccaagcccggaatgaccgttttcggcacaaa caattaaatacggtcatctgatttgtgttttttatgatttattttctgaaacgggcatgaaatttcgattattaaagtgatggtagtcacataaagtcaccttctagctaataagtgtgaccgccgtcatattacagagc gttttttatttgaaaatgaatccatgagttcatttcagacaggcaaatattcactgatatgaagcccgaactcgctggttttgcacttttgaaaacataaccgattacgtgcttaagcttctgaacctaagaggatgct SEQ ID NO.21 fuckdown aatgctgaaaacaatttcgccgttaatttctcccgaactattgaaagtgctggcagagatgggacatggagatgaaattattttttccgatgctcactttcccgcccattcgatgggaccgcaggtgatccgcgctgatggcctgttggtgagcgacttgctccaggcgattatcccgttatttgaactggacagttatgcaccgccgctggtgatgatggcggcggtagaaggtgacactctcgatcctgaagtagaacgacgttaccgtaatgcgctttcactacaagccccgtgtcctgacatcatccgcatcaatcgttttgcgttttatgaacgggcgcaaaaagcctttgcgatcgttatcacaggcgaacgagcgaagtacgggaatattcttttaaaaaaaggggtaacaccgtaatctcataccggtacgcccgcatgacgcgggcggttatcgaatgatgggg SEQ ID NO.22 wcaJup cgcaggtggcacgccggtaatgtcgaaaaccggacacgcctttattaaagaacgtatgcgcaaggaagacgccatctatggtggcgaaatgagcgcccaccattacttccgtgatttcgcttactgcgacagcggcatgatcccgtggctgctggtcgccgaactggtgtgcctgaaagataaaacgctgggcgaactggtacgcgaccggatggcggcgtttccggcaagcggtgagatcaacagcaaactggcgcaacccgttgaggcgattaaccgcgtggaacagcattttagccgtgaggcgctggcggtggatcgcaccgatggcatcagcatgacctttgccgactggcgctttaacctgcgcacctccaataccgaaccggtggtgcgcctgaatgtggaatcgcgcggtgatgtgccgctgatggaagcgcgaacgcgaactctgctgacgttgctgaacgagtaatgtcggatcttcccttaccccactgcgggtaaggggctaataacaggaacaacg SEQ ID NO.23 wcaJdown tatgagcttacgtgaaaaaaccatcagcggcgcgaagtggtcggcgattgccacggtgatcatcatcggcctcgggctggtgcagatgaccgtgctggcgcggattatcgacaaccaccagttcggcctgcttaccgtgtcgctggtgattatcgcgctggcagatacgctttctgacttcggtatcgctaactcgattattcagcgaaaagaaatcagtcaccttgaactcaccacgttgtactggctgaacgtcgggctggggatcgtggtgtgcgtggcggtgtttttgttgagtgatctcatcggcgacgtgctgaataacccggacctggcaccgttgattaaaacattatcgctggcgtttgtggtaatcccccacgggcaacagttccgcgcgttgatgcaaaaagagctggagttcaacaaaatcggcatgatcgaaaccagcgcggtgctggcgggcttcacttgtacggtggttagcgcccatttctggccgctg SEQ ID NO.24 pfkAup cgttgggtggtgcggcggacgcaaagccaggcggtgcgggctgatatgctacattaccagtctgatgttatgatgaacggcgcaattctgctggcgctggggttgtcctggtacggctggcatcgcgccgatgctctgtttgcattgggaatcggcatctatattttatatagcgcgttacgcatgggatatgaggcggtacagtcattactggatcgcgcattgcctgatgaggaacggcaagaaattattgatatcgtgacttcctggccgggtgttagcggcgctcacgatcttcgcacgcggcagtcagggccgacccgctttattcagattcatttggaaatggaagactctctgcctttggttcaggcacatatggtggcggatcaggtagagcaggctattttacggcgttttccgggatcggatgtaattatccatcaggacccctgttccgtcgtacccagggagggtaaacggtctatgctttcataatcagtataaaagagagccagacccgcattttgtgtataaaataccgccatttggcctgacctgaatcaattcagcaggaagtgattgttatactatttgcacattcgttggatcacttcgatgtgcaagaagacttccggcaacagatttcattttgcattccaaagttcagaggtagt SEQ ID NO.25 pfkAdown tgatttcggaaaaaggcagattcctttaccctgaaaccgatgacagaagcaaaaatgcctgatgcgcttcgcttatcaggcctacatgaattctgcaatttattgaatttgcaaacttttgtaggccggataaggcgttcgcgccgcatccggcatggacaaagcgcactttgtcagcaatatgaggcggatttcttccgcctttttaatccctcaacatatacccgcaagttatagccaatctttttttattctttaatgtttggttaaccttctggcacgctttgctcatcacaacacaacataagagagtcgggcgatgaacaagtggggcgtagggttaacatttttgctggcggcaaccagcgttatggcaaaggatattcagcttcttaacgtttcatatgatccaacgcgcgaattgtacgaacagtacaacaaggcattcagcgcccactggaaacagcaaactggtgataacgtggtgattcgtcagtcacacggtggctcaggtaaacaagcgacgtcggtaatcaacggtattgaagctgatgttgtcacgctggctctggcctatgacgtggacgcaattgcggaacgcgggcggattgataaag SEQ ID NO.26 pfkBup ttcagagttccatgtgacgttgtaagccaccttcagtccaaaatgttcattaatagcaacatttaatgcgctttcggagttcagcgttgtgtcttccgcgccaaagaccgaaacaccctgcgtaaatttagcgttgtcagtcaactgccatgcataagccccggaggcatagcccagcggctgggtttcactggcattgtcggtatatttgtcgtaacgcacacctggaccgaattcaaagcggaaactgtgtaccgggccattcaaaaactgacgaccataacccgcggtcaacacatcgcgctctcgatagccgttataacggtctgtcagccagcttgcctgaccaaataaatagtcataatcagttaaattaaaacggctacgcccgcccgccgcatatttttctgaagaacgctcatcattagaagaagtattactggcgttcccccacagcgaccaggcagtggtgtgtccataccaggtcatggtggtgtcagccgtaagtgaggagcttttcgtattgcctgattgtgcaagatatcctgcgttcagattaccttcgaaaggttttttagcgctggcaggatcatccatgacagtaaaaacggaatcatcggcagctgcattcagtgacgcaaacatgccccccgccaacataacgatggcaggaactgtcttcaaaagcttcatttatcaagagtccgtacaacaaaaaaagagaccatcgcggtcccggaaactttcttaaggatcaaagattagcgtccctggaaaggtaacgaattataaaaaggcgcgaataacttagcaatgtattcttatttcattttttgaataagcatgtggcgaaaacagatttttatttatatatatttatctgcaaaattttaaataaagctccaataaatcatattgttaatttcttcactttccgctgattcggtgccagactgaaatcagcctataggaggaaatg SEQ ID NO.27 pfkBdown caaaaacattcccccagcattgggggaatcatcaccaacctgtcggcaacgcgtttctccgactatgctcaaaagtcatgtgataacaaaggggtgaactatggccagtggcgatcttgtccgttatgtcataaccgtcatgttgcatgaggatacattgactgaaattaacgagttgaataattacctgactcgcgacggttttttgctcactatgacggatgatgagggaaatatccatgagctgggaactaacacttttggacttatcagtacccaaagtgaagaagaaattagagaactggtttcggggcttacccaaagtgcaaccggcaaagatcctgaaatcaccatcacgacctgggaggaatggaatagcaacagaaaataaatggtttttgggcaataatcagtctgtggtgtgcgttagctcgtgtttttacaccgcattcttgcgctaaccttatgatctggcagacaacatgggagagacatcatgtggcaggcaatcagtcgtcttttgagcgagcagttaggtgaaggcgaaatcgaactgcgtaatgaactgcctggcggagaagtccatgccgcatggcatttgcgctatgcaggacatgattttttcgtcaaatgtgatgaaagggaactgcttcccggttttaccgccgaagccgaccaactggagttactgtcgcgtagtaaaaccgtcaccgtgcctaaggtttgggcagttggcgctgaccgtgactacagttttctggtgatggattatctcccacctcgtccgctggatgcgcatagcgcatttattcttggtcagcaaattgcgcgtttacatcaatggagcgaccaaccacaatttggccttgatttcgataacgcgctctccacaactccacagcccaacacctggcaacgtcgctggtcaacgttttttgctgaacaacggat SEQ ID NO.28 wcaFup cgtctgtccgtttattttgcgaaaagcgttaaatattacgaggtaagaataagaacatgttgcttagcataatcactgtcgcgtttcgtaacctcgaagggatagtcaaaacacatgcctcgctggcgcatctggcgcaggtggaagatatcagcttcgaatggattgttgtcgatggcggttccaacgacggcacccgtgagtatctggaaaatctcaatggtatctttaacctacgctttgtcagcgagccagataacggtatctacgacgccatgaacaaaggcattgcgatggcacaaggcaagttcgcgttgtttctcaattcgggcgatatttttcatcagaatgccgcaaattttgtccgtaagttaaaaatgcaaaaagataacgtgatgatcaccggcgatgcgctgctggattttggcgacgggcataaaattaaacgtagcgccaaaccgggctggtatatttatcacagcctgcccgccagtcatcaggcgatatttttccctgtatccggcttgaaaaaatggcgttatgacctggaatataaagtttcttccgactacgcgctggcagccaaaatgtataaagccggttatgcatttaaaaaactcaatggcctggtgtctgaattttccatgggtggggtatctaccaccaataatatggaattgtgtgctgacgcgaaaaaagtccaacgacaaatattacatgtgcctggcttttgggctgaattatcctggcatttacgccaacgtactacctcaaagacgaaagccttatataacaaagtctgaatataaggaaaacca SEQ ID NO.29 wcaFdown attcaaaaatacagaggaataatacatgtcaaaagtcgctctcatcaccggtgtaaccggacaagacggttcttacctggcagagtttctgctggaaaaaggttacgaggtgcatggtattaagcgtcgcgcatcgtcattcaacaccgagcgcgtggatcacatttatcaggatccgcacacctgcaacccgaaattccatctgcattatggcgacctgagtgatacctctaacctgacgcgcattttgcgtgaagtacagccggatgaagtgtacaacctgggcgcaatgagccacgttgcggtctcttttgagtcaccagaatataccgctgacgtcgacgcgatgggtacgctgcgcctgctggaggcgatccgcttcctcggtctggaaaagaaaactcgtttctatcaggcttccacctctgaactgtatggtctggtgcaggaaattccgcagaaagagaccacgccgttctacccgcgatctccgtatgcggtcgccaaactgtacgcctactggatcaccgttaactaccgtgaatcctacggcatgtacgcctgtaacggaattctcttcaaccatgaatccccgcgccgcggcgaaaccttcgttacccgcaaaatcacccgcgcaatcgccaacatcgcccaggggctggagtcgtgcctgtacctcggcaatatggattccctgcgtg SEQ ID NO.30 zwfup taaaatcaggaaaaccgcgcgtgtccatgctgcgacagaaacgattcaccgtcggttcgctaacattggcttccagtgccatagcagcaatactcgaatggatcgcgttatcgggcgaagccagaatgacctcggcaactttgcgctctgatttgctcaaatgttccagctgagactggattttttccagcatattcatgatgtaaagagactcacgggtaatgacgatttccgcactgaaagaaatcgaaatgcagttttgtcagatattacgcctgtgtgccgtgttaatgacaaaagcagataaaaaagttgttattttttttcataacatgatcagtgtcagatttttacccaatggaaaacgatgatttttttatcagttttgccgcactttgcgcgcttttcccgtaatcgcacgggtggataagcgttt SEQ ID NO.31 zwfdown atggcggtaacgcaaacagcccaggcctgtgacctggtcattttcggcgcgaaaggcgaccttgcgcgtcgtaaattgctgccttccctgtatcaactggaaaaagccggtcagctcaacccggacacccggattatcggcgtagggcgtgctgactgggataaagcggcatataccaaagttgtccgcgaggcgctcgaaactttcatgaaagaaaccattgatgaaggtttatgggacaccctgagtgcacgtctggatttttgtaatctcgatgtcaatgacactgctgcattcagccgtctcggcgcgatgctggatcaaaaaaatcgtatcaccattaactactttgccatgccgcccagcacttttggcgcaatttgcaaagggcttggcgaggcaaaactgaatgctaaaccggcacgcgtagtcatggagaaaccgctggggacgtcgctggcgacctcgcaggaaatcaatgatcaggttggcgaatacttcgaggagtgccaggtttaccgtatcgaccactatcttggtaaagaaacgg SEQ ID NO.32 guaCup cgatgcgatagcatccggtgcgccgttattatcaatgacgtcatctgccacggcaaggcgggcttcgcgcgttgcctgagcagcaaggatttgttcgacatgctcgcgagttacatcatcgcgctgcatggtgcgcttaagttgcgtttctgggctgacatccaccacaagcactcgattcgcttttttatacagtgagttttctaccagcaatggcacaacccacagtacatagggggaagtagcttgctggatctggtgttgcgtctcttgctgaatcagcggatgcagcagggcgttaagccagtttttctcttccgggttggcgaagatccgctcgcgcaaggcccggcgctgcaatgttccatcagcagcaatcatgttagcgccaaagtgatcagcaatggcatgtagcgcaggtgcacctggttcaaccacctgacgcgcaataatatcggcatcaatgacgttaattccgagatcagcaaacgcattggcaacggtactcttgccactgccaatgcctcccgttaaggcaactatatacctcataatttatattcccgggaattcatcatgattatcaaaacgttaaaaatgagtgcacgaaagcgaaattgatgaaacgttcgctcactatttaccaggtaaatttatgggattgtagcgtaaaaaaagacaatttcgcagtcttgcgccgcattgattagtgcgtatgatagcgtcactggagttgcgctcttacccttatagccattaaccccaggaatccgcac SEQ ID NO.33 guaCdown tctcccaacgctggcgtggagcaacacgccagcgttatcccatcccactcatcgcatcgcctaaatggaaaattggcagatacattgccaccaccagcgtaccaataattcctcccgttatgatcagcaacgccggttccagtaaggctgcgaggttatccgccagcgccattgtgttttcccgatgatgatgggcgaggttgtctaacatgagatccagagagccggatgcctctcctgttctcactaattgcaaacagagcgggctaaactcaccggtattttttagcgccagccagatgggttgaccgttactgatatcgtgctggatttgtgtcagaagttgcacccagtacgggcagcgcattgtttctctgacgctctctacgccctgtaaaaaagtaatgcctgcactttgtgtcagcgccagaatcgtaaagatctgcgtgagtttttgtccccgcatcagtgaacccataatcgggatgcgtaacagcaatttctgccgcactataagccaggtcggtcggcgcatcagcaacttattggctatcgccagcagaaagccgaacaacaccagcagccagctccattcgccactaaagtctgccagcgtcatgatcccctgcgttagtgccggtag SEQ ID NO.34 BJUPP atgtctgatcgctccggcccaaaccgtttcatcattgtactgttcggtggcctgcgtccggatctggtttctccgtctcgtaccccgaacctggatcgtctgcgtcgtcgtggcgcgatgctggctcgccagcgtaccatctacccgaacgaaacgcgtgttagcctgacctccctgatcacgggtgcgaccccggatcgtcacggtatcgtgggcaaccgtttcctggaccgcgttgcagcggcgccgcgtctgatcgatactagcgatgaccgcctggtcgaagatctggacgctgcatctggtggccacctggtcggtgctccgtccctgggtgagattctggctgccaacggcaagaccttcgcagttctggcatccaacagcgcaggtgcgacgcgtatgctgaaccacaaagcgcgtagcctgagccaagtaaccctgtccggtcactttgcccgcgtagctacgtctgccgcgatgctggagcgcgttgaagcacgtctgggtccgactccagcccctaccccgcatggcactcctgacctggcggcgcaggccttcctgacctctgcgctgctggaccaggtttggccgcagatccagccggacgttgctatcctgtctttcggcgaaccggacacttcctctcatcactgcggtattggcgcaaccccgactctggaggcaatccgcttcctggaccgtcagttcggtcgcgttctggattggtgggagaccgaaggcgcaccgaagggtgtccacctgatcgtggcgtctgagcacggtcacgttaccgttcaggctcaagcggacctgctggacaccctgaaagcgaccggtctgcgctgcggtcaagtgccaggcccgggcctggacgctgtagtgatgtccggccaggtaggtgctatctacctgaccgaaccttctgaccgcgcgatccgccgtctggtggctgcgatgacggaacgtccgtggtgtggtccggttttcactgccgcgaaatctggtgaagacggcatcgccccgggttccctggcacgtcacctggttttcgccgaccacccgcgctccgcagacatcctgttttccttccgtggtgacgacggtctggactctttcggcttcccgggccgttgctggtccccgaactccccggtaggtctgggcgttcatggtggtctgcacgcaaaagaaatgtcctccctgggcatcctggctggcccgctgatccgttctggtgttgaatcccacgtcccatctggcatttgtgatttcgcgccgaccgtgctgtccctgctgggtatcagccgcccttctaccatgaccggtcgtgtgctggctgaggtcctgatcttcggcgcggaggacccaccgaccgttgaaaccgtacataaagcgcgtgcgggtggctatcgccaggacctgcgtcgcgttcaggtgggtgcaaccatctatgtagacagcgctgacgctagctcctctaaccattctacctaa SEQ ID NO.35 BJUPP MSDRSGPNRFIIVLFGGLRPDLVSPSRTPNLDRLRRRGAMLARQRTIYPNETRVSLTSLITGATPDRHGIVGNRFLDRVAAAPRLIDTSDDRLVEDLDAASGGHLVGAPSLGEILAANGKTFAVLASNSAGATRMLNHKARSLSQVTLSGHFARVATSAAMLERVEARLGPTPAPTPHGTPDLAAQAFLTSALLDQVWPQIQPDVAILSFGEPDTSSHHCGIGATPTLEAIRFLDRQFGRVLDWWETEGAPKGVHLIVASEHGHVTVQAQADLLDTLKATGLRCGQVPGPGLDAVVMSGQVGAIYLTEPSDRAIRRLVAAMTERPWCGPVFTAAKSGEDGIAPGSLARHLVFADHPRSADILFSFRGDDGLDSFGFPGRCWSPNSPVGLGVHGGLHAKEMSSLGILAGPLIRSGVESHVPSGICDFAPTVLSLLGISRPSTMTGRVLAEVLIFGAEDPPTVETVHKARAGGYRQDLRRVQVGATIYVDSADASSSNHST* SEQ ID NO.36 AMUPP atggctactacctggggcgctgctttctttatgctggtcgcaagctgcgtctgctccaccgtgttccaccgtgatcagcagacctggttcgaaggtgttttcctgagcagcatgtgcccgatcaacgtctctgccagcactctgtacggcatcatgtttgacgctggttccacgggcactcgtattcacatctacaccttcgtacagaaaatcccgggtcagctgcctatcctggaaggtgaaatcttcgagagcgtaaaaccaggtctgtccgcattcgttgaccagccgaaacaaggcgcagaaactgttgaagagctgctggaggttgctaaagattctgtcccgcgctcccactggaaacgtactcctgttgtcctgaaagcgaccgcaggcctgcgtctgctgccggaacaaaaagcagaggctctgctgttcgaggttcgtgaaattttccgtaagtctccattcctggtgccggatgattccgtgtctatcatggatggcagctatgaaggtatcctggcatgggtcacggtgaacttcctgactggccaactgcacggccactctcaaaaaaccgttggtaccctggatctgggcggcgcatctactcagatcacctttctgccgcagttcgaaaaaaccctggagcagaccccacgtggctatctgacctcttttgagatgttcaactccacgtacaaactgtacacccattcctatctgggcttcggtctgaaagctgctcgtctggcgaccctgggcgctctggaaaccgaaggtatcgacggtcatacctttcgttctgcatgcctgccgcgttggctggaggcagagtggatcttcggcggtgtaaaataccagtacggcggtaacaaggaaggtaacgaaggttctggtgaggtaggttttgagccttgttacgcggaagttctgcgtgttgttcaaggcaagctgcatcagccggatgaagtccgtaaaagctccttctacgcgttctcttattattatgaccgcgctgctgataccgacatgattgactatgaaactggcggcgtactgaaagtggaagacttcgagcgtaaagcgcgtgaagtgtgtgacaacctggaaaaattcacgagcggcagcccgttcctgtgtatggacctgtcctacattactgcactgctgaaagacggtttcggcttcgcagactctaccatcctgcagctgagcaaaaaagtgaacaacattgagactggttgggcgctgggtgcgacctttcacctgctgcagtccctgggtatctctcactaa SEQ ID NO.37 AMUPP MATTWGAAFFMLVASCVCSTVFHRDQQTWFEGVFLSSMCPINVSASTLYGIMFDAGSTGTRIHIYTFVQKIPGQLPILEGEIFESVKPGLSAFVDQPKQGAETVEELLEVAKDSVPRSHWKRTPVVLKATAGLRLLPEQKAEALLFEVREIFRKSPFLVPDDSVSIMDGSYEGILAWVTVNFLTGQLHGHSQKTVGTLDLGGASTQITFLPQFEKTLEQTPRGYLTSFEMFNSTYKLYTHSYLGFGLKAARLATLGALETEGIDGHTFRSACLPRWLEAEWIFGGVKYQYGGNKEGNEGSGEVGFEPCYAEVLRVVQGKLHQPDEVRKSSFYAFSYYYDRAADTDMIDYETGGVLKVEDFERKAREVCDNLEKFTSGSPFLCMDLSYITALLKDGFGFADSTILQLSKKVNNIETGWALGATFHLLQSLGISH* SEQ ID NO.38 yiHX atgctctatatctttgatttaggtaatgtgattgtcgatatcgactttaaccgtgtgctgggagcctggagcgatttaacgcgtattccgctggcatcgcttaagaagagttttcatatgggggaggcgtttcatcagcatgagcgtggggaaattagcgacgaagcgttcgcagaggcgctgtgtcatgagatggctctaccgctaagctacgagcagttctctcacggctggcaggcggtgtttgttgcgctgcgcccggaagtgatcgccatcatgcataaactgcgtgagcaggggcatcgcgtggtggtgctttccaataccaaccgcctgcataccaccttctggccggaagaatacccggaaattcgtgatgctgctgaccatatctatctgtcgcaagatctggggatgcgcaaacctgaagcacgaatttaccagcatgttttgcaggcggaaggtttttcacccagcgatacggtctttttcgacgataacgccgataatatagaaggagccaatcagctgggcattaccagtattctggtgaaagataaaaccaccatcccggactatttcgcgaaggtgttatgctaa SEQ ID NO.39 yiHX MLYIFDLGNVIVDIDFNRVLGAWSDLTRIPLASLKKSFHMGEAFHQHERGEISDEAFAEALCHEMALPLSYEQFSHGWQAVFVALRPEVIAIMHKLREQGHRVVVLSNTNRLHTTFWPEEYPEIRDAADHIYLSQDLGMRKPEARIYQHVLQAEGFSPSDTVFFDDNADNIEGANQLGITSILVKDKTTIPDYFAKVLC* SEQ ID NO.40 yieH atgtcccggatagaagcggtatttttcgactgcgacggtacgctggtcgacagtgaagtcatttgctctcgcgcatatgtaacgatgtttcaggaatttggtattacgctcgatcctgaagaggtattcaaacgtttcaaaggtgtaaaactgtacgaaattatcgatattgtttcccttgaacatggtgttacgttagcgaaaacagaagctgaacacgtttaccgtgcagaagtcgctcggctgttcgattcagaactggaagccatcgaaggggctggagcgctcctgtcagcgatcactgcgccaatgtgtgtggtatctaacggcccaaataacaaaatgcagcattctatgggcaagctgaatatgttgcactacttcccggataaactgttcagcggctacgatattcagcgctggaagccagacccggcgttaatgttccatgcggcaaaagcgatgaatgtaaatgtagaaaactgcattctggttgatgactcagttgccggtgcacaatctggtatcgacgcaggtatggaagtgttctacttctgcgccgacccgcacaataagccgatcgttcacccgaaagtcaccacctttacccatctttcgcagttacctgaactgtggaaagcgcgtggttgggatattacggcataa SEQ ID NO.41 yieH MSRIEAVFFDCDGTLVDSEVICSRAYVTMFQEFGITLDPEEVFKRFKGVKLYEIIDIVSLEHGVTLAKTEAEHVYRAEVARLFDSELEAIEGAGALLSAITAPMCVVSNGPNNKMQHSMGKLNMLHYFPDKLFSGYDIQRWKPDPALMFHAAKAMNVNVENCILVDDSVAGAQSGIDAGMEVFYFCADPHNKPIVHPKVTTFTHLSQLPELWKARGWDITA* SEQ ID NO.42 hxpA atgcggtgcaaaggttttctgtttgatcttgatggaacgctggtggattccctgcctgcggtagaacgggcgtggagcaactgggccagacgtcatgggttagcgccggaagaggtgctggctttcattcacggtaaacaggcgatcacctctctgcgccattttatggcgggcaaatccgaggctgatattgccgccgagtttacgcgtctggagcacatcgaggccacggaaaccgaaggtattaccgcgcttccgggggcaatcgccttactcagtcatttgaataaagcaggtattccgtgggccattgtgacttctggctccatgccggtagcgcgagcgcgccataaaatagctgggcttcccgcaccagaggtgtttgtaaccgctgagcgagtgaagcgcggaaaaccagaacctgatgcgtatctgttaggcgcgcagctgctggggcttgcgccgcaggagtgtgtggtggtggaagatgctcccgctggcgtgctttctggcctggcggcgggttgtcatgtcattgcggttaacgctccggcagataccccgcgcctgaatgaggtcgatttggtcctccacagtctggagcaaattactgtgaccaaacagccaaatggcgatgttattattcagtaa SEQ ID NO.43 hxpA MRCKGFLFDLDGTLVDSLPAVERAWSNWARRHGLAPEEVLAFIHGKQAITSLRHFMAGKSEADIAAEFTRLEHIEATETEGITALPGAIALLSHLNKAGIPWAIVTSGSMPVARARHKIAGLPAPEVFVTAERVKRGKPEPDAYLLGAQLLGLAPQECVVVEDAPAGVLSGLAAGCHVIAVNAPADTPRLNEVDLVLHSLEQITVTKQPNGDVIIQ* SEQ ID NO.44 hxpB atgtcaaccccgcgtcagattcttgctgcaatttttgatatggatggattacttatcgactcagaacctttatgggatcgagccgaactggatgtgatggcaagcctgggggtggatatctcccgtcgtaacgagctgccggacaccttaggtttacgcatcgatatggtggtcgatctttggtacgcccggcaaccgtggaatgggccaagccgtcaggaagtagtagaacgggttattgcccgtgccatttcactggttgaagagacacgtccattattaccaggcgtgcgcgaagccgttgcgttatgcaaagaacaaggtttattggtgggactggcctccgcgtcaccactacatatgctggaaaaagtgttgaccatgtttgacttacgcgacagtttcgatgccctcgcctcggccgaaaaactgccttacagcaagccgcatccgcaagtatatctcgactgcgcagcaaaactgggcgttgaccctctgacctgcgtagcgctggaagattcggtaaatggcatgatcgcctctaaagcagcccgcatgcgttccatcgtcgttcctgcgccagaagcgcaaaatgatccacgttttgtattagcagacgtcaaactttcatcgctgacagaactcaccgcaaaagaccttctcggttaa SEQ ID NO.45 hxpB MSTPRQILAAIFDMDGLLIDSEPLWDRAELDVMASLGVDISRRNELPDTLGLRIDMVVDLWYARQPWNGPSRQEVVERVIARAISLVEETRPLLPGVREAVALCKEQGLLVGLASASPLHMLEKVLTMFDLRDSFDALASAEKLPYSKPHPQVYLDCAAKLGVDPLTCVALEDSVNGMIASKAARMRSIVVPAPEAQNDPRFVLADVKLSSLTELTAKDLLG* Example 1: Construction of recombinant bacteria FUC000

[0124] The recombinant strain FUC000 was obtained by knocking out the L-fucose transporter gene encoding fucP (NCBI Gene ID 947487), the fucose isomerase gene encoding fucI (NCBI Gene ID 946195), the fucose isomerase gene encoding fucK (NCBI Gene ID 946022), and the undecylylphosphoglucose-1-phosphotransferase gene encoding wcaJ (NCBI Gene ID 946583) in Escherichia coli MC02 using the pCas9 / pTargetF gene editing system.

[0125] (1) Knock out the fucP-K gene cluster containing the three genes fucP, fucI, and fucK.

[0126] A) Based on the fucP-K gene cluster that needs to be knocked out in the host bacteria, primers fucP-up-F / fucP-up-R and fucK-down-F / fucK-down-R containing upstream and downstream homologous fragments of the fucP-K gene cluster were synthesized. Using the genome of Escherichia coli MG1655 strain as a template, the upstream and downstream target fragments were amplified respectively. After being recovered by an agarose gel extraction kit, the fucP-K up-down fragment (sequence 5'-3' is fucPup and fucKdown, respectively) was obtained by OE PCR using primers fucP-up-F / fucK-down-R.

[0127] B) Using fucI-N20-F / fucI-N20-R primers and plasmid pTargetF as a template, PCR amplification was performed to obtain the PCR product. This product replaced the N20 sequence on pTargetF with an N20 sequence complementary to the fucP-K gene cluster (ctgctgcgctttgcccgcgc), resulting in the pTargetF plasmid pTargetF-fucP-K targeting the fucP-K gene cluster. The PCR product was then used to remove the template DNA using DpnI enzyme, and transformed into *E. coli* DH5α competent cells using a heat shock transformation method. The cells were then plated on LB agar plates containing streptomycin and cultured at 37°C for plasmid extraction and sequencing.

[0128] C) Transform the pCas9 plasmid into competent cells of Escherichia coli BW25113, spread the transformed bacterial culture onto LB agar plates containing kanamycin, and incubate overnight at 37°C to obtain BW25113-pCas9.

[0129] D) Prepare BW25113-pCas9 cells as electrocompetent cells.

[0130] E) Electroporate the pTargetF-fucP-K plasmid and the fucP-K up-down fragment containing upstream and downstream homologous arms into competent E. coli BW25113-pCas9 cells, plate them on LB agar plates containing kanamycin and streptomycin, incubate at 37°C for 24 h, and perform PCR colony verification.

[0131] F) Pick the above positive clones into fresh LB liquid medium (containing a final concentration of 10 mM IPTG and 50 mg / mL Kan), and incubate at 37 ℃ for 12 h to remove the pTargetF-fucP-K plasmid, obtaining recombinant Escherichia coli BW25113∆FucI∆FucP∆FucK for the next round of knockout.

[0132] G) Eliminate pCas plasmid: Inoculate the positive clone containing the pCas plasmid into LB liquid medium and incubate overnight at 42°C to eliminate the pCas plasmid, and obtain the E. coli mutant MC02∆FucI∆FucP∆FucK.

[0133] (2) The knockout of wcaJ was performed in the same manner as described in Example 1 (1), starting from strain MC02∆FucI∆FucP∆FucK to obtain recombinant strain FUC000. The difference was that the fucP-K up-down fragment was replaced with wcaJ up-down (sequences 5'-3' are wcaJup and wcaJdown respectively). When amplifying this fragment, primers fucP-up-F, fucP-up-R, fucK-down-F, and fucK-down-R were replaced with wcaJ-up-F, wcaJ-up-R, wcaJ-down-F, and wcaJ-down-R respectively. At the same time, plasmid pTargetF-fucP-K was replaced with pTargetF-wcaJ. When preparing this plasmid, primers fucI-N20-F and fucI-N20-R were replaced with primers wcaJ-N20-F and wcaJ-N20-R respectively. Example 2 Construction of recombinant bacteria FUC001

[0134] The pCas9 / pTargetF gene editing system was used to knock out the pfkA gene (NCBI Gene ID 948412) encoding 6-phosphofructokinase 1 in E. coli FUC000, while simultaneously integrating P J23119 The promoter initiates expression of the fructose-6-phosphate isomerase-encoding gene manA (NCBI Gene ID 944840), while simultaneously integrating P... J23119 The expression of the in situ promoter gene cluster cpsG-cpsB-gmd-fcI yielded recombinant Escherichia coli FUC001, which includes the following genes: cpsB (NCBI Gene ID 946574) encoding mannose phosphate mutase, cpsG (NCBI Gene ID 946580) encoding mannose-1-phosphate guanine syltransferase, gmd (NCBI Gene ID 946562) encoding GDP-mannose 4,6-hydrolase, and fcI (NCBI Gene ID 946563) encoding GDP-L-fucose synthase.

[0135] (1) Knock out pfkA while integrating P J23119 manA gene promoter

[0136] A) Based on the host bacteria, construct a structure containing the upstream homologous arm of pfkA and P... J23119 Promoter, manA encoding gene, T rrnB The target fragments of the terminator and downstream homologous arm of pfkA were amplified using the BW25113 genome as a template with primers pfkA-up-F / pfkA-down-R. After gel extraction using an agarose gel assay kit, pfkAup-P was obtained via OE PCR. J231119 -manA-T rrnB -pfkAdown target fragment (sequences 5'-3' are: pfkBup, P) J231119 cpsG, cpsB, T rrnB 、pfkBdown).

[0137] B) Using pfkA-N20-F / pfkA-N20-R primers, the N20 sequence on pTargetF was replaced with an N20 sequence complementary to the pfkA gene, resulting in the pTargetF plasmid pTargetF-pfkA carrying the pfkA gene. The PCR product was used to remove template DNA with DpnI enzyme, and then transformed into *E. coli* DH5α competent cells using a heat shock transformation method. The cells were then plated on LB agar plates containing streptomycin and cultured at 37°C to extract the plasmid and sequence it.

[0138] C) Transform the pCas9 plasmid into competent cells of Escherichia coli FUC000, spread the transformed bacterial culture onto LB agar plates containing kanamycin, and incubate overnight at 37°C to obtain FUC000-pCas9.

[0139] D) Prepare FUC000-pCas9 cells as electrocompetent cells.

[0140] E) Combine the pTargetF-pfkA plasmid with pfkAup-P containing upstream and downstream homologous arms. J231119 The -manA-pfkAdown fragment was electroporated into competent E. coli FUC000-pCas9 cells, plated on LB agar plates containing kanamycin and streptomycin, and incubated at 37°C for 24 h for PCR colony verification.

[0141] F) Pick the above positive clones into fresh LB liquid medium (containing a final concentration of 10 mM IPTG and 50 mg / mL Kan), incubate at 37 ℃ for 12 h to remove the pTargetF-pfk plasmid, and obtain recombinant Escherichia coli FUC000ΔpfkA::P J23119 -manA is used for the next round of knockout.

[0142] G) Elimination of pCas plasmid: Positive clones containing the pCas plasmid were inoculated into LB liquid medium and cultured overnight at 42°C to eliminate the pCas plasmid, yielding the E. coli mutant FUC000ΔpfkA::P J23119 -manA.

[0143] (2) Knock out pfkB while integrating P J23119 The promoter promotes the expression of the gene cluster cpsG-cpsB in the same manner as described in Example 2 (1), from strain FUC000ΔpfkA::P J23119 -manA was used to obtain the recombinant bacteria FUC000ΔpfkA::P J23119 -manAΔpfkB::P J231119 -cpsG-cpsB. The difference is: pfkAup-P J231119 -manA-T rrnB Replace the -pfkAdown fragment with pfkBup-P J23119 -cpsG-cpsB-T rrnB -pfkBdown (sequence 5'-3' are: pfkBup, P) J23119 cpsG, cpsB, T rrnBWhen amplifying this fragment (pfkA-up-F and pfkA-down-R), replace primers pfkB-up-F and pfkB-down-R with those primers, respectively. Simultaneously, replace plasmid pTargetF-pfkA with pTargetF-pfkB. When preparing this plasmid, replace primers pfkA-N20-F and pfkA-N20-R with those primers, respectively.

[0144] (3) Knock out wcaF while integrating P J23119 The promoter promotes the expression of the gene cluster gmd-fcI in the same manner as described in Example 2 (1), from strain FUC000ΔpfkA::P J231119 -manAΔpfkB::P J23119 The recombinant strain FUC001 was obtained by starting with -cpsG-cpsB. The difference is that pfkAup-P J23119 -manA-T rrnB Replace the -pfkAdown fragment with wcaFup-P J23119 -gmd-fcI-T rrnB -wcaFdown (sequence 5'-3' are: wcaFup, P) J23119 ,gmd,fcI,T rrnB When amplifying this fragment (pfkA-up-F, pfkA-down-R), replace primers pfkA-up-F and pfkA-down-R with primers wcaF-up-F and wcaF-down-R, respectively. Simultaneously, replace plasmid pTargetF-pfkA with pTargetF-wcaF. When preparing this plasmid, replace primers pfkA-N20-F and pfkA-N20-R with primers wcaF-N20-F and wcaF-N20-R, respectively. Example 3 Construction of recombinant bacteria FUC002

[0145] Using the pCas9 / pTargetF gene editing system, P was used on the genome of E. coli FUC001. J23119 In-situ enhancement of NADP + The gene encoding glucose-6-phosphate dehydrogenase, zwf (NCBI Gene ID 948163); and the gene encoding GMP reductase, guaC (NCBI Gene ID 948986), were knocked out while integrating P. J23119 The promoter initiates the expression of the nucleoside diphosphate kinase encoding gene ndk (NCBI Gene ID 945611) and the guanosine kinase encoding gene gmk (NCBI Gene ID 948163).

[0146] (1) Replace the zwf promoter with P J23119 The promoter is the same as described in Example 2 (1), and the recombinant strain FUC001zwf::P is obtained from strain FUC001. J23119 The difference lies in: pfkAup-P J23119 -manA-T rrnB Replace the -pfkAdown fragment with zwfup-P J23119 -zwfdown (sequence 5'-3' are: zwfup, P) J23119 When amplifying this fragment, replace primers pfkA-up-F and pfkA-down-R with zwf-up-F and zwf-down-R, respectively. Simultaneously, replace plasmid pTargetF-pfkA with pTargetF-zwf. When preparing this plasmid, replace primers pfkA-N20-F and pfkA-N20-R with primers zwf-N20-F and zwf-N20-R, respectively.

[0147] (2) Knock out guaC while integrating P J23119 The promoter-activated gene cluster ndk-gmk was expressed in accordance with the method described in Example 2 (1) from strain FUC001zwf::P J231119 The recombinant strain FUC002 was obtained. The difference lies in the fact that pfkAup-P... J23119 -manA-T rrnB Replace the -pfkAdown fragment with guaCFup-P J23119 -ndk-gmk-T rrnB -guaCdown (sequence 5'-3' are: guaCup, P) J23119 ndk, gmk, T rrnB When amplifying this fragment (pfkA-up-F and pfkA-down-R), replace primers pfkA-up-F and pfkA-down-R with primers guaC-up-F and guaC-down-R, respectively. Simultaneously, replace plasmid pTargetF-pfkA with pTargetF-guaC. When preparing this plasmid, replace primers pfkA-N20-F and pfkA-N20-R with primers guaC-N20-F and guaC-N20-R, respectively.

[0148] Example 4: Recombinant strain expresses free nucleotide sugar diphosphatase and dephosphatase

[0149] Nucleotide-sugar diphosphatases from different sources with optimized synthetic codons (BJUPP from Bradyrhizobium japonicum and AMUPP from Ailuropoda melanoleuca) and dephosphatases from the same source but different origins (from Escherichia) Gene fragments of the dephosphatases yiHX, yieH, hxpA, and hxpB from *Ligusticum striatum* were ligated into the pBAD / HisB expression vector via Gibson, yielding plasmids pBAD / HisB-BJUPP-yiHX, pBAD / HisB-BJUPP-yieH, pBAD / HisB-BJUPP-hxpA, pBAD / HisB-BJUPP-hxpB, pBAD / HisB-AMUPP-yiHX, pBAD / HisB-AMUPP-yieH, pBAD / HisB-AMUPP-hxpA, and pBAD / HisB-AMUPP-hxpB. The specific steps are as follows:

[0150] Construction of recombinant plasmid pBAD / HisB-BJUPP-yiHX: The pBAD / HisB vector was amplified by PCR using primers pYB1S-F / pYB1S-R, and the vector fragment was recovered.

[0151] The artificially synthesized UPP gene fragment and PPase gene fragment (synthesized by Qingke Biotechnology) were amplified using primers pYB1S-BJUPP-F, BJUPP-R and BJUPP-yiHX-F, ​​pYB1S-yiHX-R respectively, and the target gene fragment was recovered.

[0152] The target gene fragment and the recovered vector fragment were ligated using the Gibson method (Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA, 3rd, Smith HO: Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 2009, 6:343-345.) to obtain the ligation product. The ligation product was transformed into DH5α competent cells and plated on LB agar plates containing streptomycin. After incubation at 37°C overnight, single clones were picked, plasmids were extracted, and sequencing was performed using primers pYB1S-VF and pYB1S-VR for verification. The correctly sequenced vector was named pBAD / HisB-BJUPP-yiHX.

[0153] Among them, the BJUPP gene is derived from soybean rhizobium (Bradyrhizobium japonicum), and its nucleotide sequence is shown in SEQ ID NO.34; the yiHX gene is derived from Escherichia coli, and its nucleotide sequence is shown in SEQ ID NO.38.

[0154] The sequences validated by sequencing include BJUPP and yiHX, and the other plasmids were constructed in the same manner as described above.

[0155] (4) The correctly sequenced vector pBAD / HisB-BJUPP-yiHX was transformed into the Escherichia coli mutant FUC002 by chemical transformation to obtain recombinant engineered bacteria L01, which can express nucleotide sugar diphosphatase and dephosphatase.

[0156] (5) The other plasmids carrying the genes encoding nucleotide sugar diphosphatase and dephosphatase were transferred into the substrate bacteria FUC002 in the same manner as described above to obtain engineered bacteria L02-08. Example 5: Fermentation of L-fucose by engineered strains

[0157] The L01-08 engineered strain constructed in Example 4 was: 1) inoculated into LB liquid medium and cultured overnight at 37 ℃ and 200 rpm to obtain a seed culture. The seed culture was then inoculated into 100 mL of fresh medium at a ratio of 1:100 and cultured at 37 ℃ and 200 rpm until OD reached. 600 The concentration was 0.8–1.0. IPTG was added to a final concentration of 0.2 mM, and the mixture was induced overnight for 16 h. 2) The overnight induced bacterial culture was centrifuged at 5000 rpm for 10 min to collect the cells. Simultaneously, glucose and 2 mM Mg were added to the reaction system to a final concentration of 20 g / L. 2+ 1 X M9 was reacted in a shake flask for 6 h. 1 mL of fermentation broth was taken, centrifuged at 10000 rpm for 10 min, and the supernatant was collected for HPLC analysis. The peak spectra of L-fucose and glucose standards are shown below. Figure 1-2 As shown.

[0158] The results are shown in Table 4. After fermentation, BJUPP-yieH showed a stronger ability to synthesize L-fucose compared to different nucleotide sugar diphosphatases and dephosphatases, with a higher yield of the target product L-fucose. The yield of L-fucose reached 2.84 g / L after 6 h of shake-flask culture.

[0159] Table 4. Results of L-fucose production in engineered bacteria using glucose as a substrate

[0160] Example 6: Fermentation of L-fucose by engineered strains

[0161] The L01-08 engineered strain constructed in Example 4 was: 1) inoculated into LB liquid medium and cultured overnight at 37 ℃ and 200 rpm to obtain a seed culture. The seed culture was then inoculated into 100 mL of fresh medium at a ratio of 1:100 and cultured at 37 ℃ and 200 rpm until OD reached. 600 The concentration was 0.8–1.0, and IPTG was added to a final concentration of 0.2 mM. Induction was performed overnight for 16 h. 2) The overnight induced bacterial culture was centrifuged at 5000 rpm for 10 min to collect the cells. Simultaneously, fructose and 2 mM Mg were added to the reaction system to a final concentration of 20 g / L. 2+ 1 x M9 was reacted in a shake flask for 6 h. 1 mL of fermentation broth was taken, centrifuged at 10000 rpm for 10 min, and the supernatant was collected for HPLC analysis. The peak spectra of L-fucose and fructose standards are shown below. Figure 1-2 As shown in Table 5, BJUPP-yieH exhibited a stronger ability to synthesize L-fucose, with a higher yield of the target product L-fucose. The yield of L-fucose reached 3.67 g / L after 6 h of shake-flask culture.

[0162] Table 5. Results of L-fucose production by engineered bacteria using fructose as a substrate.

[0163] Example 7: Fermentation of L-fucose by engineered strains

[0164] The L01-08 engineered strain constructed in Example 4 was: 1) inoculated into LB liquid medium and cultured overnight at 37 ℃ and 200 rpm to obtain a seed culture. The seed culture was then inoculated into 100 mL of fresh medium at a ratio of 1:100 and cultured at 37 ℃ and 200 rpm until OD reached. 600 The concentration was 0.8–1.0. IPTG was added to a final concentration of 0.2 mM, and the mixture was induced overnight for 16 h. 2) The overnight induced bacterial culture was centrifuged at 5000 rpm for 10 min to collect the cells. Simultaneously, sucrose and 2 mM Mg were added to the reaction system to a final concentration of 20 g / L. 2+ 1 X M9 was reacted in a shake flask for 6 h. 1 mL of fermentation broth was taken, centrifuged at 10000 rpm for 10 min, and the supernatant was collected for HPLC analysis. The peak spectra of L-fucose and sucrose standards are shown below. Figure 1-2 As shown in Table 6, BJUPP-yieH exhibited a stronger ability to synthesize L-fucose, with a higher yield of the target product L-fucose. The yield of L-fucose reached 3.34 g / L after 6 h of shake-flask culture.

[0165] Table 6. Results of L-fucose production by engineered bacteria using sucrose as a substrate.

[0166] Example 8: Fermentation of L-fucose by engineered strains

[0167] The L01-08 engineered strain constructed in Example 4 was: 1) inoculated into LB liquid medium and cultured overnight at 37 ℃ and 200 rpm to obtain a seed culture. The seed culture was then inoculated into 100 mL of fresh medium at a ratio of 1:100 and cultured at 37 ℃ and 200 rpm until OD reached. 600 The concentration was 0.8–1.0, and IPTG was added to a final concentration of 0.2 mM. Induction was performed overnight for 16 h. 2) The overnight induced bacterial culture was centrifuged at 5000 rpm for 10 min to collect the cells. Simultaneously, glycerol and 2 mM Mg were added to the reaction system to a final concentration of 20 g / L. 2+ 1 X M9 was reacted in a shake flask for 6 h. 1 mL of fermentation broth was taken, centrifuged at 10000 rpm for 10 min, and the supernatant was collected for HPLC analysis. The peak spectra of L-fucose and glycerol standards are shown below. Figure 1-2 As shown in Table 7, BJUPP-yieH exhibited a stronger ability to synthesize L-fucose, with a higher yield of the target product L-fucose. The yield of L-fucose reached 3.07 g / L after 6 h of shake-flask culture.

[0168] Table 7. Results of L-fucose production in engineered bacteria using glycerol as a substrate

[0169]

[0170] In summary, this invention provides a method for synthesizing L-fucose and the construction and application of recombinant Escherichia coli, which can efficiently synthesize L-fucose in a short time. The process is green, controllable, efficient and convenient, providing a new method and approach for large-scale industrial production.

[0171] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant *Escherichia coli* strain for synthesizing L-fucose, said recombinant *Escherichia coli* strain comprising at least one or more of the following modifications: (1) Introduce the nucleotide sugar diphosphatase UPP gene; (2) Introduce the dephosphatase PPase gene; (3) Enhance the expression of the glucose-6 phosphate dehydrogenase gene zwf; (4) Enhance the expression of the guanylate kinase gene gmk; (5) Enhance the expression of the nucleoside diphosphate kinase gene ndk.

2. The recombinant Escherichia coli strain according to claim 1, wherein the recombinant Escherichia coli further comprises at least one or more of the following modifications: (6) Enhance the expression of the mannose-6 phosphate isomerase gene manA; (7) Enhance the expression of the guanylate kinase gene cpsG; (8) Enhance the expression of the guanylate kinase gene cpsB; (9) Enhance the expression of the guanylate kinase gene gmd; (10) Enhance the expression of the guanylate kinase gene fcI.

3. The recombinant Escherichia coli strain according to claims 1-2, wherein the recombinant Escherichia coli strain further comprises at least one or more of the following modifications: (11) Knock out the L-fucose transporter gene fucP; (12) Knock out the L-fucose isomerase gene fucI; (13) Knock out the L-fucokinase gene fucK; (14) Knockout of the undecylylphosphoglucose-1-phosphotransferase gene wcaJ; (15) Knock out the 6-phosphofructokinase 1 gene pfkA; (16) Knock out the 6-phosphofructokinase 2 gene pfkB; (17) Knock out the clavatin biosynthesis acetyltransferase gene wcaF; (18) Knock out the GMP reductase gene guaC.

4. The recombinant Escherichia coli strain according to any one of claims 1-3, characterized in that, The enhanced expression of zwf, gmk, ndk, manA, cpsG, cpsB, gmd, and fcI is achieved through expression on chromosomes using constitutive strong promoters.

5. The recombinant Escherichia coli according to any one of claims 1-4, characterized in that, The overexpression of the nucleotide sugar diphosphatase UPP and dephosphatase PPase is achieved by using the pBAD / HisB plasmid as the expression medium.

6. The recombinant Escherichia coli according to claims 1-5, characterized in that, The nucleotide sequence of the nucleotide sugar diphosphatase UPP is shown in SEQ ID NO. 34 and 36.

7. The recombinant Escherichia coli according to claims 1-5, characterized in that, The nucleotide sequences of the dephosphatase PPase are shown in SEQ ID NO. 38, 40, 42, and 44.

8. The recombinant Escherichia coli according to claims 1-7, characterized in that, The recombinant Escherichia coli synthesizes L-fucose de novo using one of different carbon sources, such as sucrose, glucose, glycerol, or fructose, as the sole carbon source.

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