Strain with ultra-low level of endotoxin and high yield of colanic acid and use thereof

Abstract

Provided is a method for constructing a recombineered strain with a low level of endotoxin and a high yield of colanic acid, comprising: (a) constructing a colanic acid synthesis strain from a starting strain, and (b) genetically engineering, on the basis the colanic acid synthesis strain, an endotoxin synthesis pathway, wherein the recombineered strain of colanic acid comprises: a) a lipopolysaccharide core polysaccharide synthesis gene cluster comprising waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG, and waaQ on the genome being knocked out or downregulated, and the Lon protein-encoding gene lon and the HNS regulatory protein-encoding gene hns being knocked out or downregulated, b) the Vitreoscilla hemoglobin VHb being overexpressed, and c) the lpxP gene being knocked out or downregulated.

Description

A strain with ultra-low endotoxin and high production of colacid and its application

[0001] Related applications

[0002] This disclosure claims priority to international application PCT / CN2024 / 095745, filed on May 28, 2024, the entire contents of which are expressly incorporated herein by reference. Technical Field

[0003] This disclosure relates to the field of microbial technology, and in particular to a method for constructing engineered bacteria with kolanitrile acid. Background Technology

[0004] Endotoxins are a component of the outer membrane of most Gram-negative bacteria. The outer membrane is an asymmetric lipid bilayer, primarily composed of phospholipids as the inner layer and lipopolysaccharides as the outer layer. The lipopolysaccharide consists of hydrophobic lipid A, hydrophilic nonspecific core polysaccharides, and long-chain O-antigen polysaccharides. The core polysaccharide contains an exogenous hexose region and an endogenous heptose region, linked to the specific polysaccharide and lipid A, respectively. Lipid A is the key component responsible for endotoxin toxicity. Literature has reported that lipid A deficiency is lethal to Gram-negative bacteria.

[0005] Colanic acid (CA) is a bacterial extracellular polysaccharide produced by most strains of *Escherichia coli* and other species in the Enterobacteriaceae family. It is a polysaccharide synthesized by bacteria during their life processes to adapt to environmental changes and increase their survival rate. CA molecules loosely coat the bacterial cell surface, giving the cells a mucous state, preventing cell dehydration, protecting the cells, and resisting harmful substances. Under unfavorable growth conditions (e.g., dryness, low pressure, and low pH), mucous-formed strains exhibit stronger survival than wild-type strains. In 2017, Han et al. reported that feeding purified CA or CA-secreting *E. coli* significantly prolonged the lifespan of *C. elegans*. Furthermore, CA, as a unique biopolymer, possesses special biological characteristics and physiological parameters, showing broad application prospects. For example, due to its porous cellulose structure and numerous hydrophilic groups on the colloidal surface, CA is a natural hydrogel with excellent hydration capabilities and a soft texture, making it a promising candidate for future cosmetic and medical markets.

[0006] Currently, the synthesis of colacid is mainly carried out in *Escherichia coli*. For example, Sun Junsong et al. (patent CN109439708A) achieved high CA production (10.22 g / L) by transfecting an acid-fast *E. coli* with a plasmid pBhya-CAB. Wang Xiaoyuan et al. (patent CN113755515A) achieved CA synthesis of 19.79 g / L by knocking out genes related to lipopolysaccharide synthesis in *E. coli* and overexpressing genes of two precursor synthesis pathways. Shenzhen Baiyin Biotechnology Co., Ltd. reported in patent CN115287314B that a yield of 15.8 g / L was achieved through optimized fermentation of engineered *E. coli* producing colacid on a 300-L scale. All current reports on colacid synthesis are based on *Enterobacter*, but these Gram-negative bacteria contain endotoxin structures in their cell membranes. Even trace amounts of endotoxin residues in medical products can trigger strong pyrogenic reactions, greatly limiting the application of PHA in medical materials.

[0007] Currently, various medical and biological products produced based on *E. coli* require rigorous post-processing and purification steps to reduce endotoxin levels to safe levels. These steps are cumbersome and demanding, significantly increasing production costs. Research on *C. coli* (CA) is currently limited to reports on its yield, while resolving the endotoxin issue is another crucial aspect for the industrialization and application of CA. Therefore, the development of a low-endotoxin, high-yield CA synthetic strain in this paper is of great significance. It greatly simplifies post-processing and purification steps, reduces production costs, lays the foundation for the industrialization of CA, and possesses enormous market potential. Summary of the Invention

[0008] This disclosure describes the construction of a recombinant Escherichia coli strain with high CA synthesis efficiency using different strategies. Based on this strain, the endotoxin synthesis pathway was studied, and a perfect construction method was achieved by using different metabolic pathway inhibition strategies. This method reduces the endotoxin level of Escherichia coli while ensuring a high yield of colacid.

[0009] On one hand, this disclosure provides a recombinant engineered bacterium for producing colacid, wherein the recombinant engineered bacterium includes

[0010] a) The lipopolysaccharide core polysaccharide synthesis gene clusters waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG, and waaQ on the genome were knocked out or downregulated; at the same time, the Lon protein-coding gene lon and the HNS regulatory protein-coding gene hns were knocked out or downregulated;

[0011] b) Overexpression of Vibrio hygroscopicis hemoglobin VHb;

[0012] c) The lpxP gene is knocked out or downregulated.

[0013] In some specific embodiments, the recombinant engineered bacteria further include the knockout or downregulation of the kdsD gene or the eptA gene.

[0014] In some specific implementation schemes, the recombinant engineered bacteria that produce colacid include

[0015] a) The lipopolysaccharide core polysaccharide synthesis gene clusters waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG, and waaQ on the genome were knocked out or downregulated; at the same time, the Lon protein-coding gene lon and the HNS regulatory protein-coding gene hns were knocked out or downregulated;

[0016] b) Overexpression of Vibrio hygroscopicis hemoglobin VHb;

[0017] c) The lpxP gene is knocked out or downregulated;

[0018] d) The kdsD gene or eptA gene is knocked out or downregulated.

[0019] In some specific embodiments, the recombinant engineered bacteria further include the pagP gene and / or the lpxM gene being knocked out or downregulated.

[0020] In some specific implementation schemes, the recombinant engineered bacteria that produce colacid include

[0021] a) The lipopolysaccharide core polysaccharide synthesis gene clusters waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG, and waaQ on the genome were knocked out or downregulated; at the same time, the Lon protein-coding gene lon and the HNS regulatory protein-coding gene hns were knocked out or downregulated;

[0022] b) Overexpression of Vibrio hygroscopicis hemoglobin VHb;

[0023] c) The lpxP gene is knocked out or downregulated;

[0024] d) The kdsD gene or eptA gene is knocked out or downregulated;

[0025] e) The pagP gene and / or lpxM gene are knocked out or downregulated.

[0026] In some specific embodiments, the recombinant engineered bacteria further include

[0027] i) The eptA and pagP genes are knocked out or downregulated; or

[0028] ii) The eptA and lpxM genes are knocked out or downregulated; or

[0029] iii) The eptA, pagP, and lpxM genes were knocked out or downregulated.

[0030] In some specific implementation schemes, the recombinant engineered bacteria that produce colacid include

[0031] a) The lipopolysaccharide core polysaccharide synthesis gene clusters waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG, and waaQ on the genome were knocked out or downregulated; at the same time, the Lon protein-coding gene lon and the HNS regulatory protein-coding gene hns were knocked out or downregulated;

[0032] b) Overexpression of Vibrio hygroscopicis hemoglobin VHb;

[0033] c) The lpxP gene is knocked out or downregulated;

[0034] d) The eptA gene is knocked out or downregulated;

[0035] e) The pagP gene or lpxM gene is knocked out or downregulated.

[0036] In some specific implementation schemes, the recombinant engineered bacteria that produce colacid include

[0037] a) The lipopolysaccharide core polysaccharide synthesis gene clusters waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG, and waaQ on the genome were knocked out or downregulated; at the same time, the Lon protein-coding gene lon and the HNS regulatory protein-coding gene hns were knocked out or downregulated;

[0038] b) Overexpression of Vibrio hygroscopicis hemoglobin VHb;

[0039] c) The lpxP gene is knocked out or downregulated;

[0040] d) The eptA gene is knocked out or downregulated;

[0041] e) The pagP gene is knocked out or downregulated;

[0042] f) The lpxM gene is knocked out or downregulated.

[0043] In some specific embodiments, the recombinant engineered bacteria further include

[0044] Single-point mutation modification of msbAP18S or msbAP50S.

[0045] In some specific implementation schemes, the recombinant engineered bacteria that produce colacid include

[0046] a) The lipopolysaccharide core polysaccharide synthesis gene clusters waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG, and waaQ on the genome were knocked out or downregulated; at the same time, the Lon protein-coding gene lon and the HNS regulatory protein-coding gene hns were knocked out or downregulated;

[0047] b) Overexpression of Vibrio hygroscopicis hemoglobin VHb;

[0048] c) The lpxP gene is knocked out or downregulated;

[0049] d) The eptA gene is knocked out or downregulated;

[0050] e) The pagP gene is knocked out or downregulated;

[0051] f) Single-point mutation modification of msbAP18S or msbAP50S.

[0052] In some specific implementation schemes, the recombinant engineered bacteria that produce colacid include

[0053] a) The lipopolysaccharide core polysaccharide synthesis gene clusters waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG, and waaQ on the genome were knocked out or downregulated; at the same time, the Lon protein-coding gene lon and the HNS regulatory protein-coding gene hns were knocked out or downregulated;

[0054] b) Overexpression of Vibrio hygroscopicis hemoglobin VHb;

[0055] c) The lpxP gene is knocked out or downregulated;

[0056] d) The eptA gene is knocked out or downregulated;

[0057] e) The lpxM gene is knocked out or downregulated;

[0058] f) Single-point mutation modification of msbAP18S or msbAP50S.

[0059] In some specific implementation schemes, the recombinant engineered bacteria that produce colacid include

[0060] a) The lipopolysaccharide core polysaccharide synthesis gene clusters waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG, and waaQ on the genome were knocked out or downregulated; at the same time, the Lon protein-coding gene lon and the HNS regulatory protein-coding gene hns were knocked out or downregulated;

[0061] b) Overexpression of Vibrio hygroscopicis hemoglobin VHb;

[0062] c) The lpxP gene is knocked out or downregulated;

[0063] d) The eptA gene is knocked out or downregulated;

[0064] e) The pagP gene is knocked out or downregulated;

[0065] f) The lpxM gene is knocked out or downregulated;

[0066] g) Single-point mutation modification of msbAP18S or msbAP50S.

[0067] In some specific implementations, the recombinant engineered bacteria further include a knockout or downregulated lpxM gene.

[0068] In some specific implementations, the recombinant engineered bacteria further includes a knockout or downregulated lpxL gene.

[0069] In some specific implementations, the recombinant engineered bacteria further include the knockout or downregulation of the lpxM and lpxL genes.

[0070] In some specific implementation schemes, the recombinant engineered bacteria that produce colacid include

[0071] a) The lipopolysaccharide core polysaccharide synthesis gene clusters waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG, and waaQ on the genome were knocked out or downregulated; at the same time, the Lon protein-coding gene lon and the HNS regulatory protein-coding gene hns were knocked out or downregulated;

[0072] b) Overexpression of Vibrio hygroscopicis hemoglobin VHb;

[0073] c) The lpxP gene is knocked out or downregulated;

[0074] d) The eptA gene is knocked out or downregulated;

[0075] e) The pagP gene is knocked out or downregulated;

[0076] f) Single-point mutation modification of msbAP18S or msbAP50S;

[0077] g) The lpxM gene and / or lpxL gene are knocked out or downregulated.

[0078] In some specific implementation schemes, the recombinant engineered bacteria that produce colacid include

[0079] a) The lipopolysaccharide core polysaccharide synthesis gene clusters waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG, and waaQ on the genome were knocked out or downregulated; at the same time, the Lon protein-coding gene lon and the HNS regulatory protein-coding gene hns were knocked out or downregulated;

[0080] b) Overexpression of Vibrio hygroscopicis hemoglobin VHb;

[0081] c) The lpxP gene is knocked out or downregulated;

[0082] d) The eptA gene is knocked out or downregulated;

[0083] e) The lpxM gene is knocked out or downregulated;

[0084] f) Single-point mutation modification of msbAP18S or msbAP50S;

[0085] g) The lpxM gene and / or lpxL gene are knocked out or downregulated.

[0086] In some specific implementation schemes, the recombinant engineered bacteria that produce colacid include

[0087] a) The lipopolysaccharide core polysaccharide synthesis gene clusters waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG, and waaQ on the genome were knocked out or downregulated; at the same time, the Lon protein-coding gene lon and the HNS regulatory protein-coding gene hns were knocked out or downregulated;

[0088] b) Overexpression of Vibrio hygroscopicis hemoglobin VHb;

[0089] c) The lpxP gene is knocked out or downregulated;

[0090] d) The eptA gene is knocked out or downregulated;

[0091] e) The pagP gene is knocked out or downregulated;

[0092] f) The lpxM gene is knocked out or downregulated;

[0093] g) Single-point mutation modification of msbAP18S or msbAP50S;

[0094] h) The lpxM gene and / or lpxL gene are knocked out or downregulated.

[0095] In some specific embodiments, the *Vibrio hygroscopicus* hemoglobin VHb gene includes...

[0096] 1) A polynucleotide encoding SEQ ID NO: 1;

[0097] 2) A polynucleotide encoding a variant of SEQ ID NO: 1, said variant being a sequence based on amino acid point mutations, deletions, and / or additions of SEQ ID NO: 1 and having more than 90% identity with the sequence of SEQ NO: 1; and / or

[0098] 3) Encoding polynucleotides from other species that have more than 90% identity with SEQ ID NO: 1.

[0099] In some specific embodiments, the recombinant engineered bacteria are selected from one or more of the Enterobacteriaceae family, preferably Escherichia coli BL21(DE3), JM109, Nissle 1917(EcN), BW23110, or MG1655.

[0100] On the other hand, this disclosure provides the application of the recombinant engineered bacteria as described above in the preparation of products for the production of colacid.

[0101] In another aspect, this disclosure provides a method for constructing recombinant engineered bacteria for producing colacid, comprising:

[0102] a) Knock out or downregulate the lipopolysaccharide core polysaccharide synthesis gene clusters waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG, and waaQ on the genome; and simultaneously knock out or downregulate the Lon protein-coding gene lon and the HNS regulatory protein-coding gene hns.

[0103] b) Overexpression of Vibrio hygroscopicis hemoglobin VHb;

[0104] c) Knock out or downregulate the lpxP gene.

[0105] In some specific implementations, the method for constructing recombinant engineered bacteria that produce colacid further includes knocking out or downregulating the kdsD gene or the eptA gene.

[0106] In some specific implementations, the method for constructing recombinant engineered bacteria that produce colacid further includes knocking out or downregulating the pagP gene and / or the lpxM gene.

[0107] In some specific implementations, the method for constructing the recombinant engineered bacteria that produce colacid further includes

[0108] i) Knockout or downregulation of the eptA and pagP genes; or

[0109] ii) Knock out or downregulate the eptA and lpxM genes; or

[0110] iii) The eptA, pagP, and lpxM genes were knocked out or downregulated.

[0111] In some specific implementations, the method for constructing the recombinant engineered bacteria that produce colacid further includes

[0112] Single-point mutation modification of msbAP18S or msbAP50S.

[0113] In some specific implementations, the method for constructing recombinant engineered bacteria that produce colacid further includes knocking out or downregulating the lpxM gene.

[0114] In some specific implementations, the construction method of the recombinant engineered bacteria that produce colacid further includes knocking out or downregulating the lpxL gene.

[0115] In some specific implementations, the method for constructing recombinant engineered bacteria that produce colacid includes the *Vibrio hygroscopicus* hemoglobin VHb gene.

[0116] 1) A polynucleotide encoding SEQ ID NO: 1;

[0117] 2) A polynucleotide encoding a variant of SEQ ID NO: 1, said variant being a sequence based on amino acid point mutations, deletions, and / or additions of SEQ ID NO: 1 and having more than 90% identity with the sequence of SEQ NO: 1; and / or

[0118] 3) Encoding polynucleotides from other species that have more than 90% identity with SEQ ID NO: 1.

[0119] In some specific embodiments, the recombinant engineered bacteria used in the method for constructing the recombinant engineered bacteria for producing colacid are selected from one or more of the Enterobacteriaceae family, preferably Escherichia coli BL21(DE3), JM109, Nissle1917(EcN), BW23110, or MG1655.

[0120] This disclosure has the following beneficial effects:

[0121] This disclosure provides a method for constructing engineered bacteria with low endotoxin and high yield of colacid. Through the research of various engineering strategies, a colacid engineered strain that simultaneously meets the requirements of low endotoxin and high yield has been successfully constructed, laying the foundation for the industrial application of CA. Attached Figure Description

[0122] Figure 1 shows the CA yield of the lipopolysaccharide module knockout strain in Example 1.

[0123] Figure 2 shows the CA yield analysis of the lon and hns gene knockout strains in shake flasks and fermenters in Example 2.

[0124] Figure 3 shows the shake-flask level test of the vgb gene overexpressing strain in Example 3.

[0125] Figure 4 shows the fermenter level test of the vgb gene overexpression strain in Example 3.

[0126] Figure 5 shows the results of single-gene knockout of the endotoxin synthesis pathway in Example 4.

[0127] Figure 6 shows the results of dual gene knockout in the endotoxin synthesis pathway in Example 5.

[0128] Figure 7 shows the results of the three-gene and / or four-gene combination modification of the endotoxin synthesis pathway in Example 6.

[0129] Figure 8 shows the results of overexpression of the transporter protein msbA in Example 8.

[0130] Figure 9 shows the results of the lpxM gene knockout strain in Example 7 and the msbA single-point mutant strain in Example 9.

[0131] Figure 10 shows the results of the lpxM knockout strain in Example 10.

[0132] Figure 11 shows the results of the lpxL knockout strain in Example 11.

[0133] Figure 12 shows the comparison of endotoxin levels. Detailed Implementation

[0134] To make the purpose, technical solution, and effects of this disclosure clearer and more explicit, the following provides a further detailed description of this disclosure. It should be understood that the specific embodiments described herein are only for explaining this disclosure and are not intended to limit this disclosure.

[0135] Many terms and abbreviations are used in this disclosure. Definitions are provided below and should help in understanding the scope and practice of the invention.

[0136] The term "genetic modification" or "genetic combination modification" refers to the addition, deletion, or alteration of at least one DNA sequence, overexpressed protein, or combination thereof in the genome.

[0137] The term "gene knockout" refers to a technique in which exogenous DNA undergoes homologous recombination with a gene in the recipient cell's genome that has the same or similar sequence, thereby inactivating or deleting a specific gene in the body and replacing the same / similar gene sequence in the recipient cell's genome, integrating it into the recipient cell's genome.

[0138] The term "single gene knockout" refers to the complete inactivation or deletion of a single gene; the term "double gene knockout" refers to the complete inactivation or deletion of two genes; the term "triple gene knockout" refers to the complete inactivation or deletion of three genes; and the term "quadruple gene knockout" refers to the complete inactivation or deletion of four genes.

[0139] The term "overexpression" refers to the introduction of a fragment of endogenous or exogenous DNA into a microorganism to increase the intracellular activity of one or more enzymes, for example by increasing the copy number of a gene, or by using a strong promoter or a gene encoding a highly active corresponding enzyme, and optionally by a combination of these methods.

[0140] The term "single-point mutation" refers to a mutation in a single amino acid residue site or its corresponding nucleotide sequence within an amino acid sequence. It also refers to a change in a single amino acid residue site or its corresponding nucleotide within an amino acid sequence.

[0141] The term "mutation site" refers to the location of the corresponding amino acid residue in the amino acid sequence, and also includes the nucleotide base site corresponding to that amino acid residue, such as the proline at position 18 and position 50 of the msbA gene in this disclosure being mutated to serine, respectively.

[0142] The term "promoter" is a DNA sequence that binds to RNA polymerase and directs the polymerase to the correct downstream transcription start site of a polynucleotide to initiate transcription, said polynucleotide encoding a biologically active polypeptide. RNA polymerase efficiently catalyzes the assembly of messenger RNA complementary to the appropriate DNA strand of the coding region. The term "promoter" should also be understood to include a 5' non-coding region (between the promoter and the translation start point) for translation after transcription into mRNA, cis-acting transcriptional regulatory elements such as enhancers, and / or other nucleotide sequences capable of interacting with transcription factors. A promoter can be a wild-type promoter, a variant promoter, a heterozygous promoter body, or a consensus promoter.

[0143] The term "CRISPR" is a general term used to refer to three types of systems and their subtypes. CRISPR systems can be classified into Type I, Type II, and Type III, with Type II being the most commonly used gene editing tool. The CRISPR / Cas9 system is one such subtype. The CRISPR / Cas9 system consists of the Cas9 protein and gRNA. The Cas9 protein contains two main nuclease domains—the RuvC domain and the HNH domain. The RuvC domain cleaves non-complementary DNA strands, while the HNH domain cleaves complementary DNA strands. The gRNA is a chimeric RNA formed by the binding of trans-activated cripr RNA (tracrRNA) to specific cripr ribonucleic acid (crRNA), which guides Cas9 to its target. The terms "CRISPR editing technology" or "CRISPR gene editing technology" refer to gene editing using the CRISPR system, which has three most important components: crRNA, sgRNA, and the Cas9 protein.

[0144] All numerical designations used in this document, such as pH, temperature, time, concentration, content, and molecular weight, including ranges, are approximate values ​​and are varied (+) or (-) in increments of 0.1 or 1.0 where appropriate. It will be understood that all numerical designations may be preceded by the term "approximately," although not always explicitly stated.

[0145] The term “about” means within 20%, preferably within 10%, more preferably within 5%, and even more preferably within 1% of a given value or range.

[0146] As those skilled in the art will understand, for any and all purposes, particularly in providing a written description, all scopes disclosed herein also encompass any and all possible subscopes and combinations thereof. Any listed scope can be readily identified as sufficiently descriptive, and the same scope can be decomposed into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each scope discussed herein can be readily decomposed into a lower third, a middle third, and an upper third, etc.

[0147] In one aspect, this disclosure provides a recombinant engineered bacterium for producing colacid, wherein the recombinant engineered bacterium comprises

[0148] a) The lipopolysaccharide core polysaccharide synthesis gene clusters waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG, and waaQ on the genome were knocked out or downregulated; at the same time, the Lon protein-coding gene lon and the HNS regulatory protein-coding gene hns were knocked out or downregulated;

[0149] b) Overexpression of Vibrio hygroscopicis hemoglobin VHb;

[0150] c) The lpxP gene is knocked out or downregulated.

[0151] Knockout of Lipopolysaccharide (LPS) Module-Related Genes: Lipopolysaccharide is a major component of the outer membrane of most Gram-negative bacteria. LPS synthesis involves the consumption of precursors such as glucose (Glu) and galactose (Gal), which are also common raw materials for the synthesis of acetic acid (CA) polysaccharide. Therefore, knocking out the LPS synthesis pathway can redirect metabolic flux towards CA polysaccharide synthesis, increasing yield. The enzymes involved in the synthesis of core sugars in LPS are encoded by 15 genes in the waa gene cluster (waaD, waaF, waaC, waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaS, waaP, waaG, waaQ, waaA).

[0152] Knockout of the Lon and HNS genes: CA synthesis is regulated by the RcsCDB phosphate transfer system, which includes a series of genes such as rcsF, rcsC, rcsB, rcsD, and rcsA. The mechanism involves the exposed positively charged region of the RcsF protein sensing changes in cell surface environmental stress signals and transmitting this information to RcsC. The kinase activity of RcsC is activated, and the H1 domain of the histidine group undergoes autophosphorylation. The phosphate group is transferred from the Hpt domain to the D1 domain of RcsD, and the phosphorylated RcsD transfers the phosphate group to the D2 domain of the cytoplasmic protein RcsB. Phosphorylated RcsB can bind to a range of DNA molecules, thereby affecting their expression, such as cell membrane activity, motility, and extracellular polysaccharide secretion. Simultaneously, the interaction between rcsB and rcsA can significantly regulate CA synthesis. Furthermore, RcsA is rapidly degraded by the temperature-sensitive ATP-dependent protease Lon at 37°C, thus affecting CA synthesis to some extent. Hns is a protein that inhibits rcsA expression; therefore, by removing the inhibition, rcsA expression may be increased, thereby increasing CA production.

[0153] Vibrio hygroscopicus hemoglobin VHb: The gene encoding Vibrio hygroscopicus hemoglobin VHb is vgb (gene ID: L21670), and its corresponding amino acid sequence is SEQ ID NO: 1. Studies have shown that this protein can alter metabolic pathways under hypoxic conditions, improve oxygen utilization under oxygen-limited conditions, promote microbial growth, and thus enhance the synthesis of metabolites.

[0154] According to the embodiments of this disclosure, the recombinant engineered bacteria further include the knockout or downregulation of the kdsD gene or the eptA gene.

[0155] According to the embodiments of this disclosure, the recombinant engineered bacteria for producing kola sulfamate include

[0156] a) The lipopolysaccharide core polysaccharide synthesis gene clusters waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG, and waaQ on the genome were knocked out or downregulated; at the same time, the Lon protein-coding gene lon and the HNS regulatory protein-coding gene hns were knocked out or downregulated;

[0157] b) Overexpression of Vibrio hygroscopicis hemoglobin VHb;

[0158] c) The lpxP gene is knocked out or downregulated;

[0159] d) The kdsD gene or eptA gene is knocked out or downregulated.

[0160] According to embodiments of this disclosure, the recombinant engineered bacteria further include pagP gene and / or lpxM gene knocked out or downregulated.

[0161] According to the embodiments of this disclosure, the recombinant engineered bacteria for producing kola sulfamate include

[0162] a) The lipopolysaccharide core polysaccharide synthesis gene clusters waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG, and waaQ on the genome were knocked out or downregulated; at the same time, the Lon protein-coding gene lon and the HNS regulatory protein-coding gene hns were knocked out or downregulated;

[0163] b) Overexpression of Vibrio hygroscopicis hemoglobin VHb;

[0164] c) The lpxP gene is knocked out or downregulated;

[0165] d) The kdsD gene or eptA gene is knocked out or downregulated;

[0166] e) The pagP gene and / or lpxM gene are knocked out or downregulated.

[0167] According to an embodiment of this disclosure, the recombinant engineered bacteria further includes

[0168] i) The eptA and pagP genes are knocked out or downregulated; or

[0169] ii) The eptA and lpxM genes are knocked out or downregulated; or

[0170] iii) The eptA, pagP, and lpxM genes were knocked out or downregulated.

[0171] According to the embodiments of this disclosure, the recombinant engineered bacteria for producing kola sulfamate include

[0172] a) The lipopolysaccharide core polysaccharide synthesis gene clusters waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG, and waaQ on the genome were knocked out or downregulated; at the same time, the Lon protein-coding gene lon and the HNS regulatory protein-coding gene hns were knocked out or downregulated;

[0173] b) Overexpression of Vibrio hygroscopicis hemoglobin VHb;

[0174] c) The lpxP gene is knocked out or downregulated;

[0175] d) The eptA gene is knocked out or downregulated;

[0176] e) The pagP gene or lpxM gene is knocked out or downregulated.

[0177] According to the embodiments of this disclosure, the recombinant engineered bacteria for producing kola sulfamate include

[0178] a) The lipopolysaccharide core polysaccharide synthesis gene clusters waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG, and waaQ on the genome were knocked out or downregulated; at the same time, the Lon protein-coding gene lon and the HNS regulatory protein-coding gene hns were knocked out or downregulated;

[0179] b) Overexpression of Vibrio hygroscopicis hemoglobin VHb;

[0180] c) The lpxP gene is knocked out or downregulated;

[0181] d) The eptA gene is knocked out or downregulated;

[0182] e) The pagP gene is knocked out or downregulated;

[0183] f) The lpxM gene is knocked out or downregulated.

[0184] According to an embodiment of this disclosure, the recombinant engineered bacteria further includes

[0185] Single-point mutation modification of msbAP18S or msbAP50S.

[0186] Studies have shown that msbA is a lipopolysaccharide transporter that can transport the core sugar-lipid A from the inner side of the cell membrane to the outer side. Overexpression or mutation of this protein is an inhibitor of Kdo mutants, thereby enhancing the survival ability of the strain.

[0187] In this disclosure, three knockout methods were used to target the lipopolysaccharide (LPS) module genes: knockout of gene waaF, gene cluster LQ (including waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaS, waaP, waaG, waaQ), and gene cluster LG (including waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaS, waaP, waaG), resulting in three recombinant strains: ΔwaaF, ΔLG, and ΔLQ. The three recombinant strains were then validated and analyzed. The results showed that all three strains synthesized CA, with knockout of ΔLQ showing a more significant effect. Based on the ΔLQ gene, the lon and hns genes were further knocked out, resulting in a triple-knockout strain ΔLQΔlonΔhns(Δ2), which further improved the yield to 14.5 g / L. Based on the above strain Δ2, VHb protein was overexpressed using different types of promoters, and a strain with the highest CA yield was obtained and named Δ3, which achieved a CA yield of 16.8 g / L in a 5-L fermenter.

[0188] Based on the aforementioned Δ3 strain, a single-gene knockout of the key gene lpxP in the endotoxin synthesis pathway was performed, resulting in a strain named Δ3ΔlpxP. This strain produced 15.4 g / L of CA, and its endotoxin level was 10% lower than the control. Based on the Δ3ΔlpxP strain, the genes kdsD, eptA, lpxM, and lpxL were further knocked out, resulting in recombinant strains named Δ3PD, Δ3PA, Δ3PM, and Δ3PL, respectively. The CA production of Δ3PD and Δ3PA strains were 14.6 g / L and 14.2 g / L, respectively, with strain Δ3PA exhibiting even lower endotoxin levels. Based on the Δ3PA strain, the genes kdsD, pagP, lpxM, lpxL, kdsD+pagP, kdsD+lpxM, and pagP+lpxM were further knocked out, resulting in recombinant strains named Δ3PAD, Δ3PAP, Δ3PAM, Δ3PAL, Δ3PADP, Δ3PADM, and Δ3PAPM, respectively. The CA yields of strains Δ3PAD, Δ3PAP, and Δ3PADP were not significantly affected. The endotoxin level of strain Δ3PAP decreased significantly, while the endotoxin levels of the other two strains increased. The yield of strain Δ3PAM reached the same level as the original strain at 48h, and its endotoxin level also decreased significantly.

[0189] Based on strains Δ3PADP and Δ3PADM, msbA was overexpressed using three different promoter strengths (P12 / P18 / P20, low, medium, and high). The results showed that msbA overexpression could significantly improve the growth performance of the strains.

[0190] The strain Δ3PAP was selected as the chassis strain for further modification. Using CRISPR editing technology, amino acid P (proline) at position 18 or amino acid P (serine) in the msbA gene was mutated to S (serine), resulting in two strains named Δ3PAP18 and Δ3PAP50, respectively. Both strains showed a significant reduction in endotoxin levels, while yield was also somewhat affected, but their survival ability was improved to a certain extent. Based on these two strains (Δ3PAP18 and Δ3PAP50), the lpxM gene was further knocked out to obtain strains Δ3PAP18M and Δ3PAP50M. Based on the above strains, the lpxL gene was further knocked out to obtain strains Δ3PAP18ML and Δ3PAP50ML. The yield of these two strains was further improved compared to strains Δ3PAP18 and Δ3PAP50, their growth status was better, and their endotoxin levels were further reduced.

[0191] According to the embodiments of this disclosure, the recombinant engineered bacteria for producing kola sulfamate include

[0192] a) The lipopolysaccharide core polysaccharide synthesis gene clusters waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG, and waaQ on the genome were knocked out or downregulated; at the same time, the Lon protein-coding gene lon and the HNS regulatory protein-coding gene hns were knocked out or downregulated;

[0193] b) Overexpression of Vibrio hygroscopicis hemoglobin VHb;

[0194] c) The lpxP gene is knocked out or downregulated;

[0195] d) The eptA gene is knocked out or downregulated;

[0196] e) The pagP gene is knocked out or downregulated;

[0197] f) Single-point mutation modification of msbAP18S or msbAP50S.

[0198] According to the embodiments of this disclosure, the recombinant engineered bacteria for producing kola sulfamate include

[0199] a) The lipopolysaccharide core polysaccharide synthesis gene clusters waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG, and waaQ on the genome were knocked out or downregulated; at the same time, the Lon protein-coding gene lon and the HNS regulatory protein-coding gene hns were knocked out or downregulated;

[0200] b) Overexpression of Vibrio hygroscopicis hemoglobin VHb;

[0201] c) The lpxP gene is knocked out or downregulated;

[0202] d) The eptA gene is knocked out or downregulated;

[0203] e) The lpxM gene is knocked out or downregulated;

[0204] f) Single-point mutation modification of msbAP18S or msbAP50S.

[0205] According to the embodiments of this disclosure, the recombinant engineered bacteria for producing kola sulfamate include

[0206] a) The lipopolysaccharide core polysaccharide synthesis gene clusters waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG, and waaQ on the genome were knocked out or downregulated; at the same time, the Lon protein-coding gene lon and the HNS regulatory protein-coding gene hns were knocked out or downregulated;

[0207] b) Overexpression of Vibrio hygroscopicis hemoglobin VHb;

[0208] c) The lpxP gene is knocked out or downregulated;

[0209] d) The eptA gene is knocked out or downregulated;

[0210] e) The pagP gene is knocked out or downregulated;

[0211] f) The lpxM gene is knocked out or downregulated;

[0212] g) Single-point mutation modification of msbAP18S or msbAP50S.

[0213] According to embodiments of this disclosure, the recombinant engineered bacteria further include a knocked-out or downregulated lpxM gene.

[0214] According to embodiments of this disclosure, the recombinant engineered bacteria further include a knocked-out or downregulated lpxL gene.

[0215] According to embodiments of this disclosure, the recombinant engineered bacteria further include the knockout or downregulation of the lpxM and lpxL genes.

[0216] According to the embodiments of this disclosure, the recombinant engineered bacteria for producing kola sulfamate include

[0217] a) The lipopolysaccharide core polysaccharide synthesis gene clusters waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG, and waaQ on the genome were knocked out or downregulated; at the same time, the Lon protein-coding gene lon and the HNS regulatory protein-coding gene hns were knocked out or downregulated;

[0218] b) Overexpression of Vibrio hygroscopicis hemoglobin VHb;

[0219] c) The lpxP gene is knocked out or downregulated;

[0220] d) The eptA gene is knocked out or downregulated;

[0221] e) The pagP gene is knocked out or downregulated;

[0222] f) Single-point mutation modification of msbAP18S or msbAP50S;

[0223] g) The lpxM gene and / or lpxL gene are knocked out or downregulated.

[0224] According to the embodiments of this disclosure, the recombinant engineered bacteria for producing kola sulfamate include

[0225] a) The lipopolysaccharide core polysaccharide synthesis gene clusters waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG, and waaQ on the genome were knocked out or downregulated; at the same time, the Lon protein-coding gene lon and the HNS regulatory protein-coding gene hns were knocked out or downregulated;

[0226] b) Overexpression of Vibrio hygroscopicis hemoglobin VHb;

[0227] c) The lpxP gene is knocked out or downregulated;

[0228] d) The eptA gene is knocked out or downregulated;

[0229] e) The lpxM gene is knocked out or downregulated;

[0230] f) Single-point mutation modification of msbAP18S or msbAP50S;

[0231] g) The lpxM gene and / or lpxL gene are knocked out or downregulated.

[0232] According to the embodiments of this disclosure, the recombinant engineered bacteria for producing kola sulfamate include

[0233] a) The lipopolysaccharide core polysaccharide synthesis gene clusters waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG, and waaQ on the genome were knocked out or downregulated; at the same time, the Lon protein-coding gene lon and the HNS regulatory protein-coding gene hns were knocked out or downregulated;

[0234] b) Overexpression of Vibrio hygroscopicis hemoglobin VHb;

[0235] c) The lpxP gene is knocked out or downregulated;

[0236] d) The eptA gene is knocked out or downregulated;

[0237] e) The pagP gene is knocked out or downregulated;

[0238] f) The lpxM gene is knocked out or downregulated;

[0239] g) Single-point mutation modification of msbAP18S or msbAP50S;

[0240] h) The lpxM gene and / or lpxL gene are knocked out or downregulated.

[0241] According to embodiments of this disclosure, the *Vibrio hygroscopicus* hemoglobin VHb gene includes...

[0242] 1) A polynucleotide encoding SEQ ID NO: 1;

[0243] 2) A polynucleotide encoding a variant of SEQ ID NO: 1, said variant being a sequence based on amino acid point mutations, deletions, and / or additions of SEQ ID NO: 1 and having more than 90% identity with the sequence of SEQ NO: 1; and / or

[0244] 3) Encoding polynucleotides from other species that have more than 90% identity with SEQ ID NO: 1.

[0245] According to the embodiments of this disclosure, the recombinant engineered bacteria are selected from one or more of the Enterobacteriaceae family, preferably Escherichia coli BL21(DE3), JM109, Nissle 1917(EcN), BW23110, or MG1655.

[0246] Another aspect of this disclosure provides the application of the recombinant engineered bacteria as described above in the preparation of products for the production of colacid.

[0247] One aspect of this disclosure provides a method for constructing a recombinant engineered bacterium for producing colacid, comprising:

[0248] a) Knock out or downregulate the lipopolysaccharide core polysaccharide synthesis gene clusters waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG, and waaQ on the genome; and simultaneously knock out or downregulate the Lon protein-coding gene lon and the HNS regulatory protein-coding gene hns.

[0249] b) Overexpression of Vibrio hygroscopicis hemoglobin VHb;

[0250] c) Knock out or downregulate the lpxP gene.

[0251] According to the embodiments of this disclosure, the method for constructing recombinant engineered bacteria that produce colacid further includes knocking out or downregulating the kdsD gene or the eptA gene.

[0252] According to the embodiments of this disclosure, the method for constructing recombinant engineered bacteria that produce colacid further includes knocking out or downregulating the pagP gene and / or the lpxM gene.

[0253] According to the embodiments of this disclosure, the method for constructing recombinant engineered bacteria for producing colacid further includes:

[0254] i) Knockout or downregulation of the eptA and pagP genes; or

[0255] ii) Knock out or downregulate the eptA and lpxM genes; or

[0256] iii) The eptA, pagP, and lpxM genes were knocked out or downregulated.

[0257] According to the embodiments of this disclosure, the method for constructing recombinant engineered bacteria for producing colacid further includes:

[0258] Single-point mutation modification of msbAP18S or msbAP50S.

[0259] According to the embodiments of this disclosure, the method for constructing recombinant engineered bacteria that produce colacid further includes knocking out or downregulating the lpxM gene.

[0260] According to the embodiments of this disclosure, the method for constructing recombinant engineered bacteria that produce colacid further includes knocking out or downregulating the lpxL gene.

[0261] According to the embodiments of this disclosure, the *Vibrio hygroscopicus* hemoglobin VHb gene mentioned in the method for constructing recombinant engineered bacteria that produce colacid includes...

[0262] 1) A polynucleotide encoding SEQ ID NO: 1;

[0263] 2) A polynucleotide encoding a variant of SEQ ID NO: 1, said variant being a sequence based on amino acid point mutations, deletions, and / or additions of SEQ ID NO: 1 and having more than 90% identity with the sequence of SEQ NO: 1; and / or

[0264] 3) Encoding polynucleotides from other species that have more than 90% identity with SEQ ID NO: 1.

[0265] According to the embodiments of this disclosure, the recombinant engineered bacteria used in the method for constructing the recombinant engineered bacteria for producing colacid are selected from one or more of the Enterobacteriaceae family, preferably Escherichia coli BL21(DE3), JM109, Nissle1917(EcN), BW23110, or MG1655.

[0266] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Nothing in this disclosure should be construed as an admission that the embodiments described herein are not entitled to precede such disclosure by any prior invention.

[0267] Embodiments are also provided in which any of the embodiments described herein may be combined with any or more of the other embodiments, unless otherwise stated, and provided that such combinations are not mutually exclusive.

[0268] The technical solutions provided in this disclosure will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of this disclosure.

[0269] Materials and Methods

[0270] Seed culture medium:

[0271] Yeast powder 5-10g / L, yeast peptone 5-10g / L, sodium chloride 5-10g / L

[0272] Fermentation medium:

[0273] Yeast extract 10-20 g / L, yeast peptone 20-40 g / L, KH₂PO₄ 2-4 g / L, K₂HPO₄ 10-15 g / L, glucose 10-30 g / L, pH 4.5-7.5

[0274] Molecular cloning related reagents:

[0275] Seamless cloning enzyme (Takara), DNA polymerase (Takara), DNA recovery kit (Shanghai Sangon Biotech), plasmid extraction kit (Shanghai Sangon Biotech), DNA marker (Takara)

[0276] Seed culture methods:

[0277] Using an inoculation loop, pick up a single colony streaked on a plate and inoculate it into 3-7 mL of seed culture medium. Incubate at 34-38℃ for 10-18 h.

[0278] Shake-flask fermentation test culture method:

[0279] Take the seed liquid and inoculate it into the fermentation medium at an inoculation rate of 1%-10%. Place the inoculated fermentation medium at 25-32℃ and incubate for 24-48 hours.

[0280] Polysaccharide detection methods:

[0281] (1) Sample preparation:

[0282] After culturing for 48 hours, the fermentation broth was centrifuged at 10,000-15,000 rpm for 20 minutes in a refrigerated centrifuge, and the supernatant was collected. 1.5-4 times the volume of anhydrous ethanol was added to the supernatant, and the mixture was allowed to stand overnight at 2-8°C. The ethanol-precipitated solution was centrifuged at high speed for 20 minutes, the supernatant was discarded, and the precipitate was retained. An appropriate amount of sterile water was added to reconstitute the precipitate, and the resulting solution was the extracted polysaccharide extract.

[0283] (2) Carbazole method for detecting CA yield:

[0284] Colanic acid (CA) is an acidic polysaccharide composed of D-glucose, L-fucose, D-galactose, and D-glucuronic acid. The polysaccharide hydrolyzes with a sulfuric acid solution containing sodium tetraborate at high temperature. The hydrolysis product, D-glucuronic acid, undergoes a condensation reaction with carbazole reagent to produce a purple-red compound. The color intensity of this compound is linearly related to its content within a certain range. The content of uronic acid can be calculated using a colorimetric method, thus yielding the CA content.

[0285] Example

[0286] Example 1: Construction of a single-gene knockout strain of the lipopolysaccharide pathway

[0287] Lipopolysaccharide (LPS) synthesis consumes precursor substances and competes with the CA synthesis pathway; therefore, gene (cluster) knockout in this pathway is considered. The gene cluster waaF (gene ID: 948135), gene cluster LG (containing genes waaL (gene ID: 948148), waaU (gene ID: 948147), waaZ (gene ID: 948146), waaY (gene ID: 948145), waaR (gene ID: 948142), waaO (gene ID: 948143), waaB (gene ID: 948144), waaS (gene ID: 948151), waaP (gene ID: 948150), and waaG (gene ID: 948150) were knocked out, respectively. 49)) or gene cluster LQ (waaL (gene ID: 948148), waaU (gene ID: 948147), waaZ (gene ID: 948146), waaY (gene ID: 948145), waaR (gene ID: 948142), waaO (gene ID: 948143), waaB (gene ID: 948144), waaS (gene ID: 948151), waaP (gene ID: 948150), waaG (gene ID: 948149), waaQ (gene ID: 948155)). The genome was knocked out using CRISPR-Cas9.

[0288] 1. Construction of homologous arm fragments and sgRNA plasmids

[0289] To perform gene knockout, the corresponding 1000bp nucleotide sequences upstream and downstream of the gene (cluster) were first amplified by PCR and named waaF-arm1, waaF-arm2; LG-arm1, LG-arm2; and LQ-arm1, LQ-arm2, respectively. The two corresponding upstream and downstream homologous arms were then ligated into a single fragment waaF, LG-arm, or LQ-arm using fusion PCR. Next, to construct the sgRNA, the corresponding gene sequence was used as a template, and the sgRNA sequence was designed online using the Chopchop website. The resulting sequence was ligated into the pTarget plasmid, yielding plasmids containing the corresponding gene sgRNA, namely pTarget-waaF, pTarget-LG, and pTarget-LQ.

[0290] 2. Preparation of competent colon cells

[0291] To perform gene knockout, the constructed homologous arm fragment and sgRNA plasmid need to be introduced into the cells. For this purpose, the cells need to be made into competent cells that can absorb foreign DNA. (1) Inoculate the strain containing pCas9 plasmid into 50ml LB medium at an inoculation rate of 1% to 2% and incubate at 30℃. When the OD600 reaches 0.1 to 0.2, add 2mL of 1M L-arabinose (final concentration 40mM) and incubate at 30℃ to induce the expression of recombinase. The induction time is at least 1h. When the OD600 reaches 0.6 to 0.7, start to make competent cells. (2) Collect the bacterial cells by centrifugation at 5000rpm for 5min at 4℃, add pre-cooled sterile 10% glycerol to resuspend the bacterial cells, and wash 3 times (wash with 1mL glycerol solution). (3) Finally, add 500uL of pre-cooled 10% glycerol (50mL bacterial solution concentrated 100 times) to prepare competent cells. Aliquot 90uL per tube and freeze at -80℃ for later use.

[0292] 3. Electrical knockout

[0293] The obtained homologous arm fragments waaF-arm, LG-arm, LQ-arm and sgRNA plasmids pTarget-waaF, pTarget-LG, pTarget-LQ were added to competent cells in a ratio of 1:1 to 1:4, respectively. The cells were gently mixed and incubated on ice for 30 min. The mixture was then transferred to an electroporation cuvette (1 mm cuvette, 1800 V, 200 Ω). Immediately after electroporation, 600 μL of pre-chilled LB medium was added, and the mixture was transferred to a 1.5 mL EP tube and incubated at 30°C for 2.5–3 h. The cells were then plated onto plates containing spectinomycin and kanamycin resistance, incubated at 30°C, and PCR was performed to verify the knockout results after bacterial growth.

[0294] 4. Plasmid elimination

[0295] The selected strains with successful deletion were inoculated into 2 mL of LB solution (with corresponding addition of antibiotic Kan), and IPTG was added to a final concentration of 0.5 mM. The mixture was then incubated at 30°C for 12 h to eliminate the pTarget plasmid. A small amount of bacterial culture was streaked onto a Kan plate and incubated at 30°C. Single colonies that grew were then picked and spotted onto both Spc and Kan plates. Strains that grew on Kan plates but not on Spc plates were those with eliminated pTarget plasmids. Single colonies were inoculated onto LB medium and incubated overnight at 37°C (or 42°C) (high temperature causes loss of the temperature-sensitive pCas plasmid). A small amount of bacterial culture was then streaked onto an antibiotic-free plate and incubated at 37°C. Single colonies were spotted onto both Kan and antibiotic-free plates and incubated at 37°C. Colonies that grew on antibiotic-free plates but not on Kan plates were those with successfully eliminated plasmids. Finally, strains ΔF, ΔLG, and ΔLQ with the relevant gene knocked out were obtained.

[0296] The knockout strains ΔF, ΔLG, and ΔLQ were inoculated into seed culture medium and then transferred to fermentation medium for fermentation testing. The fermentation broth was centrifuged at high speed, and the supernatant was collected. The yields of the three strains were detected by a colorimetric method, showing values ​​of 0.3 g / L, 0.15 g / L, and 0.4 g / L, respectively (Figure 1). These results indicate that knocking out genes related to the lipopolysaccharide pathway significantly promotes CA synthesis, with the highest yield observed when the LQ gene cluster was knocked out.

[0297] Example 2: Knockout of lon and hns genes

[0298] Using the same techniques as in Example 1, homologous arms hns-arm and lon-arm, and sgRNA plasmids pTarget-hns and pTarget-lon, respectively, with hns (gene ID: 945829) and lon (gene ID: 945085) knocked out, were constructed based on the ΔLQ strain. The competent cell preparation-electroporation knockout-plasmid loss steps were performed in the same manner, sequentially knocking out the lon and hns genes to obtain the recombinant strain Δ2. Analysis and verification of this strain in shake flasks and 5-L fermenters showed yields of 2.2 g / L and 14.5 g / L, respectively, as shown in Figure 2, representing a significant increase compared to the control strain.

[0299] Example 3: Overexpression of VHb hemoglobin from *Vibrio hystericus*

[0300] The hemoglobin VHb encoding gene, vgb, was ligated into four different promoters, pET, Pvgb, Ptac, and aP4 (promoter sequences SEQ ID NO: 2–SEQ ID NO: 5, respectively). The resulting recombinant plasmids were then transformed into strain Δ2, yielding recombinant strains named Δ2 / pET-vgb, Δ2 / Pvgb-vgb, Δ2 / Ptac-vgb, and Δ2 / aP4-vgb, respectively. Yields of the four strains were analyzed at both shake-flask and 5-L fermenter levels, as shown in Figures 3 and 4. The results showed that strain Δ2 / Ptac-vgb (renamed Δ3) exhibited the highest yields at both shake-flask and fermenter levels, achieving a CA yield of 16.8 g / L in the 5-L fermenter, a 15.9% increase compared to the control CA.

[0301] Example 4: Single gene knockout of the endotoxin synthesis pathway

[0302] Based on the aforementioned Δ3 strain, single-gene knockouts of the key genes lpxL, lpxP, and lpxM in the endotoxin synthesis pathway were performed. After multiple attempts, only lpxP was successfully knocked out, and this strain was named Δ3ΔlpxP. Knockouts of the other two genes in strain Δ3 were attempted to induce lethality, but no corresponding mutant strains could be obtained. Yield analysis in a 5-L fermenter showed that the CA yield of the Δ3ΔlpxP strain was 15.4 g / L, and its growth OD600 was even higher than that of the control strain (Figure 5a); the endotoxin level was reduced by 10% compared to the control (Figure 5b). These results indicate that knocking out the lpxP gene does not affect the growth of the strain and plays a certain role in reducing endotoxin levels, but the effect of single-gene knockout on endotoxin reduction is very limited.

[0303] Example 5: Dual gene knockout of the endotoxin synthesis pathway

[0304] Based on the Δ3ΔlpxP strain, the genes kdsD, eptA, lpxM, and lpxL were further knocked out, resulting in recombinant strains named Δ3PD, Δ3PA, Δ3PM, and Δ3PL, respectively. Strains Δ3PM and Δ3PL could not be successfully constructed; therefore, knocking out lpxM or lpxL on the Δ3ΔlpxP strain resulted in its lethality. As shown in Figure 6, the performance of the Δ3PD and Δ3PA strains was analyzed and compared in a 5-L fermenter. Their CA yields were 14.6 g / L and 14.2 g / L, respectively, slightly lower than the control strain's yield (16.8 g / L). The endotoxin levels synthesized by the strains were detected; the endotoxin levels of Δ3PD and Δ3PA were 85.0% and 70.0% of the control, respectively, indicating a further effective reduction in endotoxin levels.

[0305] Example 6: Construction of strains modified with three and / or four gene combinations

[0306] Based on the Δ3PA strain, the genes kdsD, pagP, lpxM, lpxL, kdsD+pagP, and kdsD+lpxM were further knocked out, resulting in recombinant strains named Δ3PAD, Δ3PAP, Δ3PAM, Δ3PAL, Δ3PADP, and Δ3PADM, respectively. The results are shown in Figure 7a. Strains Δ3PAD, Δ3PAP, and Δ3PADP showed good growth, not significantly different from the control, and some even outperformed the control, such as Δ3PADP. Figure 7b shows that the corresponding CA yields were not significantly affected, with 24h yields of 15.3 g / L, 16.6 g / L, and 14.6 g / L, respectively. However, strains Δ3PAM and Δ3PADM showed significantly slower growth compared to the original strain, and their CA yields were somewhat affected. An exception was strain Δ3PAM, which showed slow growth and a reduced 24h yield, but its 48h yield still reached the same level as the original strain, at 16.8 g / L.

[0307] Analysis of endotoxin levels revealed that only strains Δ3PAP and Δ3PAM showed a significant decrease in endotoxin levels among the various knockout strains constructed, at 9.5% and 42.5% of the control strains, respectively. This means that the endotoxin levels of strains Δ3PAP and Δ3PAM were reduced by 90.5% and 57.5% compared to the control, respectively. Conversely, the endotoxin levels of recombinant strains constructed using other knockout methods all showed varying degrees of increase (as shown in Figure 7c).

[0308] The strain Δ3PAL could not be successfully constructed; knocking out the lpxL gene led to the lethality of the strain.

[0309] Example 7: Analysis of lpxM gene knockout strains

[0310] The endotoxin levels of the constructed strains Δ3PAP and Δ3PAM were significantly reduced. Further engineering strategies were combined, with the lpxM gene knocked out from the Δ3PAP strain to obtain the recombinant strain Δ3PAPM. Analysis of this strain and the control strain Δ3 under the same conditions revealed that the growth performance of the recombinant strain was generally poor, with a very low OD600, as shown in Figure 9a. Endotoxin level analysis showed a significant reduction in endotoxin levels in the constructed Δ3PAPM strain, decreasing by 99.0% compared to the control strain (Figure 9b).

[0311] Example 8: Overexpression of transport protein msbA

[0312] In the above study, the endotoxin level of strain Δ3PAPM was significantly reduced, but the growth of the strain was significantly affected. Further research is needed on how to maintain low endotoxin levels while ensuring the normal growth of the strain.

[0313] The fast-growing strain Δ3PADP and the slow-growing strain Δ3PADM were selected. The msbA protein was overexpressed using three different promoter intensities (P12 / P18 / P20, low, medium, and high) to investigate the effects of different expression intensities on endotoxin and CA production levels. As shown in Figure 8a, the results indicate that overexpression of msbA at a certain intensity significantly improves the growth performance of the strains (e.g., strain Δ3PADM / P18-msbA), while other intensities do not necessarily improve growth performance. Figure 8b shows that CA production levels increased (e.g., strain Δ3PADM / P20-msbA) or decreased (e.g., strain Δ3PADP / P12-msbA); endotoxin levels, however, increased to varying degrees compared to the control (Figure 8c). It is impossible to obtain a strain that simultaneously achieves low endotoxin levels and high CA production through overexpression of msbA at different intensities.

[0314] Example 9: Single-point mutation of transport protein msbA

[0315] Analysis of Examples 6 and 7 showed that strains Δ3PAP and Δ3PAPM could significantly reduce endotoxin levels. However, the growth rate of strain Δ3PAPM became very slow. To ensure normal growth and further strain modification, it was necessary to enhance the growth capacity of the strain. Strain Δ3PAP was selected as the chassis strain for further modification using CRISPR technology (operation as described above).

[0316] Example 1) The 18th amino acid P (proline) or the 50th amino acid P in the msbA gene of the genome was mutated to S (serine), resulting in two strains named Δ3PAP18 and Δ3PAP50, respectively. As shown in Figure 9, the growth status of the two strains Δ3PAP18 and Δ3PAP50 was restored to the same level as the control strain, and the endotoxin levels were significantly reduced compared to the control, only 10% of the control strain. The corresponding yields were 14.01 g / L and 11.4 g / L, respectively.

[0317] Example 10 lpxM gene knockout

[0318] The aforementioned studies have shown that knockout of lpxM has a positive effect on reducing endotoxin levels, but knockout of this gene significantly affects the activity of the strain and the yield of CA (Figure 9b). Strains Δ3PAP18 and Δ3PAP50, obtained through single-point mutation of msbA, showed improved survival to some extent. Further knockout of the lpxM gene on these two strains yielded strains Δ3PAP18M and Δ3PAP50M. Validation analysis of these two strains in a 5-L fermenter showed good growth, with corresponding yields of 12.4 g / L and 15.9 g / L, respectively (Figure 10).

[0319] Example 11 lpxL gene knockout

[0320] In previous studies, direct knockout of the lpxL gene in strains resulted in bacterial death. Here, we further knocked out the lpxL gene in strains Δ3PAP18M and Δ3PAP50M without causing bacterial death, successfully obtaining strains Δ3PAP18ML and Δ3PAP50ML. As shown in Figure 11, these two strains were validated and analyzed in a 5-L fermenter. Both strains showed good growth, even superior to their counterparts Δ3PAP18M and Δ3PAP50M, with yields of 18.1 g / L and 18.3 g / L, respectively. The yields of these two strains were actually further improved compared to before the lpxL gene knockout.

[0321] Example 12: Comparative Analysis of Endotoxin Levels

[0322] The endotoxin levels of the recombinant strains Δ3PAP18M, Δ3PAP50M, Δ3PAP18ML, and Δ3PAP50ML obtained above were compared and analyzed with the control strain, and the results are shown in Figure 12. The results showed that the endotoxin levels of these four strains were significantly reduced compared with the control. Among them, strains Δ3PAP18ML and Δ3PAP50ML had the lowest endotoxin levels, at 18.5 EU / mg and 500 EU / mg, respectively, which were 99.9% lower than the endotoxin levels of the control strain. The yields of these two strains are shown in Figure 11, reaching 18.1 g / L and 18.3 g / L, respectively. Through a series of engineering strategies, strains that can simultaneously achieve low endotoxin levels and high yields of colacid were finally obtained.

[0323] Table 1 Sequence Information

[0324] Table 2 Primer sequence list

[0325] It should be understood that the application of this disclosure is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

[0326] By incorporating via reference

[0327] The full contents of every patent and scientific document mentioned in this article are incorporated herein by reference for all purposes.

[0328] Equivalence

[0329] This invention may be embodied in other specific ways without departing from its spirit or essential characteristics. Therefore, the above embodiments should be considered illustrative in all cases, and not as limiting of the invention described herein. Consequently, the scope of the invention is defined by the appended claims rather than by the foregoing description, and is intended to be encompassed by all variations within the equivalent meaning and scope of the claims.

Claims

1. A recombinant engineered bacterium for producing colacid, wherein, The recombinant engineered bacteria include a) The lipopolysaccharide core polysaccharide synthesis gene clusters waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG, and waaQ on the genome were knocked out or downregulated; at the same time, the Lon protein-coding gene lon and the HNS regulatory protein-coding gene hns were knocked out or downregulated; b) Overexpression of Vibrio hygroscopicis hemoglobin VHb; c) The lpxP gene is knocked out or downregulated.

2. The recombinant engineered bacteria as described in claim 1, wherein, The recombinant engineered bacteria further include the knockout or downregulation of the kdsD gene or the eptA gene.

3. The recombinant engineered bacteria as described in claim 2, wherein, The recombinant engineered bacteria further include pagP gene and / or lpxM gene knocked out or downregulated.

4. The recombinant engineered bacteria as described in claim 1, wherein, The recombinant engineered bacteria further include i) The eptA and pagP genes were knocked out or downregulated; ii) The eptA and lpxM genes are knocked out or downregulated; or iii) The eptA, pagP, and lpxM genes were knocked out or downregulated.

5. The recombinant engineered bacteria as described in claim 4, wherein, The recombinant engineered bacteria further include Single-point mutation modification of msbAP18S or msbAP50S.

6. The recombinant engineered bacteria as described in claim 5, wherein, The recombinant engineered bacteria further include strains in which the lpxM gene has been knocked out or downregulated.

7. The recombinant engineered bacteria as described in claim 6, wherein, The recombinant engineered bacteria further include strains in which the lpxL gene has been knocked out or downregulated.

8. The recombinant engineered bacteria as described in claim 1, wherein, The Vibrio hygroscopic hemoglobin VHb gene includes 1) A polynucleotide encoding SEQ ID NO: 1; 2) A polynucleotide encoding a variant of SEQ ID NO: 1, said variant being a sequence based on amino acid point mutations, deletions, and / or additions of SEQ ID NO: 1 and having more than 90% identity with the sequence of SEQ NO: 1; and / or 3) Encoding polynucleotides from other species that have more than 90% identity with SEQ ID NO:

1.

9. The recombinant engineered bacteria as described in claim 1, wherein, The recombinant engineered bacteria are selected from one or more of the Enterobacteriaceae family, preferably Escherichia coli BL21(DE3), JM109, Nissle 1917(EcN), BW23110, or MG1655.

10. The use of the recombinant engineered bacteria as described in any one of claims 1-9 in the preparation of products that produce colacid.

11. A method for constructing a recombinant engineered bacterium for producing colacid, comprising: a) Knock out or downregulate the lipopolysaccharide core polysaccharide synthesis gene clusters waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG, and waaQ on the genome; and simultaneously knock out or downregulate the Lon protein-coding gene lon and the HNS regulatory protein-coding gene hns. b) Overexpression of Vibrio hygroscopicis hemoglobin VHb; c) Knock out or downregulate the lpxP gene.

12. The method of claim 11, wherein, Further measures include knocking out or downregulating the kdsD gene or the eptA gene.

13. The method of claim 12, wherein, Further measures include knocking out or downregulating the pagP gene and / or the lpxM gene.

14. The method of claim 11, wherein, Further including i) Knockout or downregulation of the eptA and pagP genes; or ii) Knock out or downregulate the eptA and lpxM genes; or iii) The eptA, pagP, and lpxM genes were knocked out or downregulated.

15. The method of claim 14, wherein, Further including Single-point mutation modification of msbAP18S or msbAP50S.

16. The method of claim 15, wherein, Further measures include knocking out or downregulating the lpxM gene.

17. The method of claim 16, wherein, Further measures include knocking out or downregulating the lpxL gene.

18. The method of claim 11, wherein, The Vibrio hygroscopic hemoglobin VHb gene includes 1) A polynucleotide encoding SEQ ID NO: 1; 2) A polynucleotide encoding a variant of SEQ ID NO: 1, said variant being a sequence based on amino acid point mutations, deletions, and / or additions of SEQ ID NO: 1 and having more than 90% identity with the sequence of SEQ NO: 1; and / or 3) Encoding polynucleotides from other species that have more than 90% identity with SEQ ID NO:

1.

19. The method of claim 11, wherein, The recombinant engineered bacteria are selected from one or more of the Enterobacteriaceae family, preferably Escherichia coli BL21(DE3), JM109, Nissle 1917(EcN), BW23110, or MG1655.

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