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

Ultralow endotoxin colanic acid high-yield strain and application thereof

[0001] Related applications

[0002] The present disclosure claims priority to International Application PCT / CN2024 / 095745 filed on May 28, 2024, the entire contents of which are hereby expressly incorporated by reference in their entirety. TECHNICAL FIELD

[0003] The present disclosure relates to the field of microbiology, in particular to a method for constructing an engineered colanic acid producing strain. BACKGROUND

[0004] Endotoxin is a component of the outer membrane of most Gram-negative bacteria, which is an asymmetric lipid bilayer mainly composed of phospholipids as the inner layer and lipopolysaccharides as the outer layer. Lipopolysaccharides are composed of hydrophobic lipid A, hydrophilic non-specific core polysaccharides, and long-chain O-antigen polysaccharides. The core polysaccharides contain an outer hexose region and an inner heptose region, which are connected to specific polysaccharides and lipid A, respectively. Lipid A is the key component that causes endotoxin toxicity. It has been reported that the absence of lipid A is fatal to Gram-negative bacteria.

[0005] Colanic acid (CA) is a bacterial exopolysaccharide produced by most E. coli strains and other species of Enterobacteriaceae. It is a polysaccharide synthesized by bacteria during life activities to adapt to environmental changes and improve their survival rate. CA molecules loosely wrap around the surface of the bacteria, making the bacteria appear in a mucous state, preventing cell dehydration, protecting cells, and resisting harmful substances. In unfavorable environmental conditions (such as dryness, low pressure, and low pH), mucous strains have stronger survivability than wild-type strains. In 2017, Han et al. reported that feeding purified CA or E. coli that can secrete CA can significantly prolong the lifespan of Caenorhabditis elegans. In addition, CA, as a unique active biopolymer, has special biological characteristics and physiological parameters, and has wide application prospects. For example, due to its porous cellulose structure and large number of hydrophilic groups on the colloid surface, CA is a natural hydrogel with excellent water replenishing ability and soft texture, making it a good candidate product for future cosmetic and medical markets.

[0006] Current synthesis of colanic acid is mainly in Escherichia coli. For example, Sun Junsong et al. (patent CN109439708A) realized high yield of CA by an acid-resistant E. coli, which transfected a plasmid pBhya-CAB in its intracellular to achieve a yield of 10.22 g / L, Wang Xiaoyuan et al. (patent CN113755515A) knocked out the genes related to synthesis of lipopolysaccharide of E. coli and overexpressed two genes of precursor synthesis pathway, finally realized synthesis of 19.79 g / L CA. Shenzhen Bailing Biotechnology Co., Ltd. reported in patent CN115287314B that through optimization of 300-L scale fermentation of engineered E. coli producing colanic acid, a yield of 15.8 g / L was achieved. All the reports of synthesis of colanic acid are based on Enterobacteriaceae, but the endotoxin structure exists in the cell membrane of this kind of gram-negative bacteria, and trace residues of endotoxin in medical products will cause strong pyrogen reaction, which greatly limits the application of PHA in medical materials.

[0007] Currently, various medical biological products produced based on E. coli need to undergo strict post-processing purification steps to reduce endotoxin to a safe level, which is tedious and harsh, and greatly increases the production cost of the product. The research on CA is currently limited to the report of its yield, and the endotoxin problem of CA needs to be solved before it goes to industrialization and product application. Therefore, the CA synthesis strain developed by the present disclosure has great significance, greatly simplifies the post-processing purification steps, reduces the production cost, lays a foundation for the industrialization of CA, and has great market prospect. SUMMARY

[0008] The present disclosure constructs a recombinant E. coli with high efficiency of CA synthesis by adopting different strategies, and then studies the synthesis pathway of endotoxin based on the strain, realizes a perfect construction method through different metabolic pathway inhibition strategies, reduces the endotoxin level of E. coli, and ensures the high yield of colanic acid.

[0009] In one aspect, the present disclosure provides a recombinant engineering bacterium for producing colanic acid, wherein the recombinant engineering bacterium comprises

[0010] a) the lipopolysaccharide core polysaccharide synthesis gene cluster waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG and waaQ on the genome are knocked out or down-regulated; and the Lon protein coding gene lon and the HNS regulatory protein coding gene hns are knocked out or down-regulated;

[0011] b) overexpressing the hemoglobin VHb of Vitreoscilla;

[0012] c) the lpxP gene is knocked out or down-regulated.

[0013] In some embodiments, the recombinant engineered bacteria further comprises a kdsD gene or an eptA gene being knocked out or down-regulated.

[0014] In some embodiments, the recombinant engineered bacteria for producing colanic acid comprises

[0015] a) the lipopolysaccharide core polysaccharide synthesis gene cluster waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG and waaQ on the genome are knocked out or down-regulated; meanwhile, the Lon protein encoding gene Ion and the HNS regulatory protein encoding gene hns are knocked out or down-regulated;

[0016] b) the Vitreoscilla hemoglobin VHb is overexpressed;

[0017] c) the lpxP gene is knocked out or down-regulated;

[0018] d) the kdsD gene or the eptA gene is knocked out or down-regulated.

[0019] In some embodiments, the recombinant engineered bacteria further comprises a pagP gene and / or a lpxM gene being knocked out or down-regulated.

[0020] In some embodiments, the recombinant engineered bacteria for producing colanic acid comprises

[0021] a) the lipopolysaccharide core polysaccharide synthesis gene cluster waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG and waaQ on the genome are knocked out or down-regulated; meanwhile, the Lon protein encoding gene Ion and the HNS regulatory protein encoding gene hns are knocked out or down-regulated;

[0022] b) the Vitreoscilla hemoglobin VHb is overexpressed;

[0023] c) the lpxP gene is knocked out or down-regulated;

[0024] d) the kdsD gene or the eptA gene is knocked out or down-regulated.

[0025] e) the pagP gene and / or the lpxM gene is knocked out or down-regulated.

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

[0027] i) the eptA gene and the pagP gene are knocked out or down-regulated; or

[0028] ii) the eptA gene and the lpxM gene are knocked out or down-regulated; or

[0029] iii) the eptA gene, the pagP gene and the lpxM gene are knocked out or down-regulated.

[0030] In some embodiments, the recombinant engineered bacteria for producing colanic acid comprise

[0031] a) the lipopolysaccharide core polysaccharide synthesis gene cluster waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG and waaQ on the genome are knocked out or down-regulated; meanwhile the Lon protein encoding gene Ion and the HNS regulatory protein encoding gene hns are knocked out or down-regulated;

[0032] b) the Vitreoscilla hemoglobin VHb is overexpressed;

[0033] c) the lpxP gene is knocked out or down-regulated;

[0034] d) the eptA gene is knocked out or down-regulated;

[0035] e) the pagP gene or the lpxM gene is knocked out or down-regulated.

[0036] In some embodiments, the recombinant engineered bacteria for producing colanic acid comprise

[0037] a) the lipopolysaccharide core polysaccharide synthesis gene cluster waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG and waaQ on the genome are knocked out or down-regulated; meanwhile the Lon protein encoding gene Ion and the HNS regulatory protein encoding gene hns are knocked out or down-regulated;

[0038] b) the Vitreoscilla hemoglobin VHb is overexpressed;

[0039] c) the lpxP gene is knocked out or down-regulated;

[0040] d) the eptA gene is knocked out or down-regulated;

[0041] e) the pagP gene is knocked out or down-regulated;

[0042] f) the lpxM gene is knocked out or down-regulated.

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

[0044] a single point mutation modification of msbAP18S or msbAP50S.

[0045] In some embodiments, the recombinant engineered bacteria for producing colanic acid comprise

[0046] a) the lipopolysaccharide core polysaccharide synthesis gene cluster waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG and waaQ on the genome are knocked out or down-regulated; meanwhile, the Lon protein encoding gene Ion and the HNS regulatory protein encoding gene hns are knocked out or down-regulated;

[0047] b) overexpression of Vitreoscilla hemoglobin VHb;

[0048] c) the lpxP gene is knocked out or down-regulated;

[0049] d) the deptA gene is knocked out or down-regulated;

[0050] e) the pagP gene is knocked out or down-regulated;

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

[0052] In some embodiments, the recombinant engineered bacteria for producing colanic acid comprise

[0053] a) the lipopolysaccharide core polysaccharide synthesis gene cluster waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG and waaQ on the genome are knocked out or down-regulated; meanwhile, the Lon protein encoding gene Ion and the HNS regulatory protein encoding gene hns are knocked out or down-regulated;

[0054] b) overexpression of Vitreoscilla hemoglobin VHb;

[0055] c) the lpxP gene is knocked out or down-regulated;

[0056] d) the deptA gene is knocked out or down-regulated;

[0057] e) the lpxM gene is knocked out or down-regulated;

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

[0059] In some embodiments, the recombinant engineered bacteria for producing colanic acid comprise

[0060] a) the lipopolysaccharide core polysaccharide synthesis gene cluster waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG and waaQ on the genome are knocked out or down-regulated; meanwhile, the Lon protein encoding gene Ion and the HNS regulatory protein encoding gene hns are knocked out or down-regulated;

[0061] b) overexpression of Vitreoscilla hemoglobin VHb;

[0062] c) the lpxP gene is knocked out or down-regulated;

[0063] d) the deptA gene is knocked out or down-regulated;

[0064] e) the pagP gene is knocked out or down-regulated;

[0065] f) the lpxM gene is knocked out or down-regulated;

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

[0067] In some embodiments, the recombinant engineered bacteria further comprise the lpxM gene is knocked out or down-regulated.

[0068] In some embodiments, the recombinant engineered bacteria further comprise the lpxL gene is knocked out or down-regulated.

[0069] In some embodiments, the recombinant engineered bacteria further comprise the lpxM gene and the lpxL gene are knocked out or down-regulated.

[0070] In some embodiments, the recombinant engineered bacteria for producing colanic acid comprise

[0071] a) the lipopolysaccharide core polysaccharide synthesis gene cluster waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG and waaQ on the genome are knocked out or down-regulated; and the Lon protein encoding gene Ion and the HNS regulatory protein encoding gene hns are knocked out or down-regulated;

[0072] b) overexpressing Vitreoscilla hemoglobin VHb;

[0073] c) the lpxP gene is knocked out or down-regulated;

[0074] d) the deptA gene is knocked out or down-regulated;

[0075] e) the pagP gene is knocked out or down-regulated;

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

[0077] g) the lpxM gene and / or the lpxL gene are knocked out or down-regulated.

[0078] In some embodiments, the recombinant engineered bacteria for producing colanic acid comprise

[0079] a) the lipopolysaccharide core polysaccharide synthesis gene cluster waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG and waaQ on the genome are knocked out or down-regulated; meanwhile, the Lon protein encoding gene Ion and the HNS regulatory protein encoding gene hns are knocked out or down-regulated;

[0080] b) overexpression of Vitreoscilla hemoglobin VHb;

[0081] c) the lpxP gene is knocked out or down-regulated;

[0082] d) the deptA gene is knocked out or down-regulated;

[0083] e) the lpxM gene is knocked out or down-regulated;

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

[0085] g) the lpxM gene and / or the lpxL gene is knocked out or down-regulated.

[0086] In some embodiments, the recombinant engineered bacteria producing carboxylic acids include

[0087] a) the lipopolysaccharide core polysaccharide synthesis gene cluster waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG and waaQ on the genome are knocked out or down-regulated; meanwhile, the Lon protein encoding gene Ion and the HNS regulatory protein encoding gene hns are knocked out or down-regulated;

[0088] b) overexpression of Vitreoscilla hemoglobin VHb;

[0089] c) the lpxP gene is knocked out or down-regulated;

[0090] d) the deptA gene is knocked out or down-regulated;

[0091] e) the pagP gene is knocked out or down-regulated;

[0092] f) the lpxM gene is knocked out or down-regulated;

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

[0094] h) the lpxM gene and / or the lpxL gene is knocked out or down-regulated.

[0095] In some embodiments, the Vitreoscilla 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, which is a sequence based on the amino acid point mutations, deletions and / or additions of SEQ ID NO: 1 and which has more than 90% identity to the sequence of SEQ ID NO: 1; and / or

[0098] 3) a polynucleotide encoding a sequence from other species which has more than 90% identity to SEQ ID NO: 1.

[0099] In some 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] In another aspect, the present disclosure provides use of the recombinant engineered bacteria as described above in the preparation of a product producing colanic acid.

[0101] In yet another aspect, the present disclosure provides a method for constructing a recombinant engineered bacteria producing colanic acid, comprising:

[0102] a) knocking out or down the genes of lipopolysaccharide core polysaccharide synthesis gene cluster waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG and waaQ on the genome; and knocking out or down the genes of Lon protein encoding gene lon and HNS regulatory protein encoding gene hns;

[0103] b) overexpressing the Vitreoscilla hemoglobin VHb;

[0104] c) knocking out or down the lpxP gene.

[0105] In some embodiments, the method for constructing a recombinant engineered bacteria producing colanic acid further comprises knocking out or down the kdsD gene or the eptA gene.

[0106] In some embodiments, the method for constructing a recombinant engineered bacteria producing colanic acid further comprises knocking out or down the pagP gene and / or the lpxM gene.

[0107] In some embodiments, the method for constructing a recombinant engineered bacteria producing colanic acid further comprises

[0108] i) knocking out or down the eptA gene and the pagP gene; or

[0109] ii) knocking out or down the eptA gene and the lpxM gene; or

[0110] iii) the eptA gene, the pagP gene and the lpxM gene are knocked out or down-regulated.

[0111] In some specific embodiments, the method for constructing the recombinant engineered bacteria for producing colanic acid further comprises

[0112] single point mutation modification of msbAP18S or msbAP50S.

[0113] In some specific embodiments, the method for constructing the recombinant engineered bacteria for producing colanic acid further comprises knocking out or down-regulating the lpxM gene.

[0114] In some specific embodiments, the method for constructing the recombinant engineered bacteria for producing colanic acid further comprises knocking out or down-regulating the lpxL gene.

[0115] In some specific embodiments, the method for constructing the recombinant engineered bacteria for producing colanic acid, the Vitreoscilla hemoglobin VHb gene comprises

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

[0117] 2) a polynucleotide encoding a variant of SEQ ID NO: 1, which is a sequence based on the amino acid point mutation, deletion and / or addition of SEQ ID NO: 1 and has more than 90% identity with the sequence of SEQ ID NO: 1; and / or

[0118] 3) a polynucleotide encoding a sequence from other species with more than 90% identity with SEQ ID NO: 1.

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

[0120] The present disclosure has the following beneficial effects:

[0121] The present disclosure provides a method for constructing low-endotoxin and high-yield colanic acid engineering bacteria, which successfully constructs colanic acid engineering bacteria strains that meet the requirements of low endotoxin and high yield through the research of various engineering strategies, and lays a foundation for the industrialization application of CA. BRIEF DESCRIPTION OF DRAWINGS

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

[0123] Figure 2 is the CA yield analysis of the lon and hns gene knockout strain in Example 2.

[0124] Figure 3 is a shake flask level test of vgb gene overexpression strain in Example 3.

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

[0126] Figure 5 is the result of single gene knockout of endotoxin synthesis pathway in Example 4.

[0127] Figure 6 is the result of double gene knockout of endotoxin synthesis pathway in Example 5.

[0128] Figure 7 is the result of triple gene and / or quadruple gene combination modification of endotoxin synthesis pathway in Example 6.

[0129] Figure 8 is the result of transporter protein msbA overexpression in Example 8.

[0130] Figure 9 is the result of lpxM gene knockout strain in Example 7, msbA single point mutation strain in Example 9.

[0131] Figure 10 is the result of lpxM knockout strain in Example 10.

[0132] Figure 11 is the result of lpxL knockout strain in Example 11.

[0133] Figure 12 is the comparison of endotoxin level analysis. DETAILED DESCRIPTION

[0134] In order to make the purpose, technical solutions and effects of the present disclosure clearer and more explicit, the present disclosure is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present disclosure and do not limit the present disclosure.

[0135] In the present disclosure, many terms and abbreviations are used. Definitions are provided below, which should help to understand the scope and practice of the present invention.

[0136] The term "genetic modification" or "genetic combination modification" refers to adding, deleting or changing at least one DNA sequence in the genome, overexpressing proteins and combinations thereof.

[0137] The term "gene knockout" means that in the art, exogenous DNA homologous recombination occurs with genes having the same or similar sequences in the genome of the recipient cell, so that the specific genes of the organism are inactivated or deleted, thereby replacing the same / similar gene sequence in the genome of the recipient cell, and integrating into the genome of the recipient cell.

[0138] The term "single gene knockout" means that a single gene is completely inactivated or deleted; the term "double gene knockout" means that both of the two genes are completely inactivated or deleted; the term "triple gene knockout" means that all of the three genes are completely inactivated or deleted; and the term "four gene knockout" means that all of the four genes are completely inactivated or deleted.

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

[0140] The term "single point mutation" means a mutation in an amino acid residue site in an amino acid sequence or a change in an amino acid residue or its corresponding nucleotide in an amino acid residue site in an amino acid sequence.

[0141] The term "mutation site" means a site corresponding to an amino acid residue in an amino acid sequence, and also includes a site corresponding to a nucleotide base of the amino acid residue, for example, the proline at the 18th position of the msbA gene or the proline at the 50th position of the msbA gene is mutated to serine.

[0142] The term "promoter" is a DNA sequence that binds RNA polymerase and directs the polymerase to the correct downstream transcription start site of a polynucleotide encoding a polypeptide having a biological activity to initiate transcription. The RNA polymerase effectively 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 the 5' non-coding region (between the promoter and the translation start site) 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. The promoter can be a wild-type promoter, a variant promoter, a hybrid promoter, or a consensus promoter.

[0143] The term "CRISPR" is a general term applied to three types of systems and system subtypes. CRISPR systems can be divided into type I, type II, type III, among which type II system is the most commonly used gene editing tool, and CRISPR / Cas9 system belongs to one of them. CRISPR / Cas9 system is composed of Cas9 protein and gRNA, Cas9 protein contains two main nuclease domains, RuvC domain and HNH domain, the former cuts the non-complementary DNA strand, and the latter cuts the complementary DNA strand; gRNA is a chimeric RNA formed by the combination of trans-activated crispr RNA (tracrRNA) and specific crispr ribonucleic acid (crRNA), which is used to guide Cas9 to its target. The term "CRISPR editing technology" or "CRISPR gene editing technology" means that gene editing is achieved by using CRISPR system, and there are three most important components crRNA, sgRNA and Cas9 protein.

[0144] All numerical designations used herein, such as pH, temperature, time, concentration, content and molecular weight, including ranges, are approximations, and are varied by + or - 0.1 or 1.0 as appropriate. It will be understood that the term "about" can be preceded by the term "approximately" although it is not always expressly stated. It will be understood that the term "about" can be preceded by the term "approximately" although it is not always expressly 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 will be understood by those skilled in the art, for any and all purposes, particularly in providing a written description, all ranges disclosed herein are also intended to encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same. The same can be considered to have explicitly shown other possible sub-ranges and / or the combination of sub-ranges thereof. By way of non-limiting example, each range discussed herein can be easily recognized as sufficiently describing and enabling the same. The same can be considered to have explicitly shown other possible sub-ranges and / or the combination of sub-ranges thereof.

[0147] In one aspect of the present disclosure, a recombinant engineering bacterium for producing colanic acid is provided, wherein the recombinant engineering bacterium comprises

[0148] a) the gene clusters waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG and waaQ on the genome are knocked out or down-regulated; meanwhile, the Lon protein coding gene lon and the HNS regulatory protein coding gene hns are knocked out or down-regulated;

[0149] b) overexpressing Vitreoscilla hemoglobin VHb;

[0150] c) the lpxP gene is knocked out or down-regulated.

[0151] Lipopolysaccharide module related gene knockout: Lipopolysaccharide is the main component of the outer membrane of most gram-negative bacteria. During the synthesis of lipopolysaccharide, the consumption of precursors such as glucose (Glu) and galactose (Gal) is involved, and these precursors are also common raw materials for the synthesis of CA polysaccharide. Therefore, by knocking out the lipopolysaccharide synthesis pathway, the metabolic flow can be directed to the synthesis of CA polysaccharide, thereby improving the yield. The enzymes involved in the synthesis of the core sugar in the lipopolysaccharide are encoded by 15 genes (waaD, waaF, waaC, waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaS, waaP, waaG, waaQ, and waaA) in the waa gene cluster.

[0152] lon and hns gene knockout: The synthesis of CA is regulated by the RcsCDB phosphotransfer system, which includes a series of genes such as rcsF, rcsC, rcsB, rcsD, and rcsA. The principle is that the positively charged region of the RcsF protein can sense changes in the cell surface environmental stress signal and transmit the information to RcsC. The kinase activity of RcsC is activated, and the H1 domain of the histidine group is autophosphorylated. The phosphate group is transferred from the Hpt domain to the D1 domain of RcsD. Phosphorylated RcsD transfers the phosphate group to the D2 domain of the cytoplasmic protein RcsB. Phosphorylated RcsB can bind to a series of DNA, thereby affecting their expression, such as the bacterial membrane, motility, and exopolysaccharide secretion. At the same time, the interaction of rcsB and rcsA can significantly regulate the synthesis of CA. In addition, RcsA is rapidly degraded by the temperature-sensitive ATP-dependent protease Lon at 37°C, thereby affecting the synthesis of CA to some extent. Hns is a protein that inhibits the expression of rcsA, so by removing the inhibition, the expression of rcsA can be increased, thereby increasing the yield of CA.

[0153] Vitreoscilla hemoglobin VHb: The Vitreoscilla hemoglobin VHb encoding gene is vgb (Gene ID: L21670), and the corresponding amino acid sequence is SEQ ID ID NO: 1. Studies have shown that this protein can change the metabolic pathway under low oxygen conditions, improve oxygen utilization under limited dissolved oxygen conditions, promote microbial growth, and thereby improve the synthesis of metabolic products.

[0154] According to the embodiments of the present disclosure, the recombinant engineering bacteria for producing carboxyamidotriazole produce further include that the kdsD gene or the eptA gene is knocked out or down-regulated.

[0155] According to the embodiments of the present disclosure, the recombinant engineering bacteria for producing carboxyamidotriazole produce further include

[0156] a) the lipopolysaccharide core polysaccharide synthesis gene cluster waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG and waaQ on the genome are knocked out or down-regulated; meanwhile, the Lon protein encoding gene lon and the HNS regulatory protein encoding gene hns are knocked out or down-regulated;

[0157] b) the Vitreoscilla hemoglobin VHb is overexpressed;

[0158] c) the lpxP gene is knocked out or down-regulated;

[0159] d) the kdsD gene or the eptA gene is knocked out or down-regulated.

[0160] According to embodiments of the present disclosure, the recombinant engineering bacteria further include that the pagP gene and / or the lpxM gene is knocked out or down-regulated.

[0161] According to embodiments of the present disclosure, the recombinant engineering bacteria for producing colanic acid include

[0162] a) the lipopolysaccharide core polysaccharide synthesis gene cluster waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG and waaQ on the genome are knocked out or down-regulated; meanwhile, the Lon protein encoding gene lon and the HNS regulatory protein encoding gene hns are knocked out or down-regulated;

[0163] b) the Vitreoscilla hemoglobin VHb is overexpressed;

[0164] c) the lpxP gene is knocked out or down-regulated;

[0165] d) the kdsD gene or the eptA gene is knocked out or down-regulated;

[0166] e) the pagP gene and / or the lpxM gene is knocked out or down-regulated.

[0167] According to embodiments of the present disclosure, the recombinant engineering bacteria further include

[0168] i) the eptA gene and the pagP gene are knocked out or down-regulated; or

[0169] ii) the eptA gene and the lpxM gene are knocked out or down-regulated; or

[0170] iii) the eptA gene, the pagP gene and the lpxM gene are knocked out or down-regulated.

[0171] According to embodiments of the present disclosure, the recombinant engineering bacteria for producing colanic acid include

[0172] a) the lipopolysaccharide core polysaccharide synthesis gene cluster waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG and waaQ on the genome are knocked out or down-regulated; meanwhile, the Lon protein encoding gene lon and the HNS regulatory protein encoding gene hns are knocked out or down-regulated;

[0173] b) the Vitreoscilla hemoglobin VHb is overexpressed;

[0174] c) the lpxP gene is knocked out or down-regulated;

[0175] d) the deptA gene is knocked out or down-regulated;

[0176] e) the pagP gene or the lpxM gene is knocked out or down-regulated.

[0177] According to an embodiment of the present disclosure, the recombinant engineering bacteria for producing capuramycin include

[0178] a) the lipopolysaccharide core polysaccharide synthesis gene cluster waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG and waaQ on the genome are knocked out or down-regulated; meanwhile, the Lon protein encoding gene lon and the HNS regulatory protein encoding gene hns are knocked out or down-regulated;

[0179] b) the Vitreoscilla hemoglobin VHb is overexpressed;

[0180] c) the lpxP gene is knocked out or down-regulated;

[0181] d) the deptA gene is knocked out or down-regulated;

[0182] e) the pagP gene is knocked out or down-regulated;

[0183] f) the lpxM gene is knocked out or down-regulated.

[0184] According to an embodiment of the present disclosure, the recombinant engineering bacteria further include

[0185] a single point mutation modification of msbAP18S or msbAP50S.

[0186] Studies have shown that msbA is a lipopolysaccharide transporter protein that can transport the core sugar-lipid A on the inner side of the cell membrane to the outer side of the cell membrane, and overexpression or mutation of the protein is an inhibitor of Kdo mutant, thereby strengthening the survival ability of the strain.

[0187] Three kinds of knockouts were made in the disclosure for lipopolysaccharide module genes: genes 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) were knocked out respectively to obtain three recombinant bacteria AwaaF, ALG and ALQ; verification analysis was performed on the three recombinant bacteria respectively. It was found that the three bacteria all had CA synthesis, and the effect of knockout ALQ was more obvious. On the basis of the above ALQ, the genes lon and hns were further knocked out respectively to obtain a triple-knockout strain ALQAlonAhns (Δ2), and the yield of the strain was further improved to 14.5 g / L. On the basis of the strain Δ2, VHb protein was overexpressed by different types of promoters, and a strain with the highest CA yield was screened and named as Δ3, and the CA yield of the strain in a 5-L fermenter reached 16.8 g / L.

[0188] On the basis of the above strain Δ3, single gene knockout was performed on the key gene lpxP of the endotoxin synthesis pathway, and the strain was named as Δ3AlpxP. The CA yield of the strain was 15.4 g / L, and the endotoxin level was reduced by 10% compared with the control. On the basis of the strain Δ3AlpxP, the genes kdsD, eptA, lpxM, and lpxL were further knocked out respectively, and the corresponding recombinant strains were named as Δ3PD, Δ3PA, Δ3PM, and Δ3PL respectively. Among them, the CA yields of the strains Δ3PD and Δ3PA were 14.6 g / L and 14.2 g / L respectively, and the endotoxin level of the strain Δ3PA was lower. On the basis of the strain Δ3PA, the genes kdsD, pagP, lpxM, lpxL, kdsD+pagP, kdsD+lpxM, and pagP+lpxM were further knocked out respectively, and the corresponding recombinant strains were named as Δ3PAD, Δ3PAP, Δ3PAM, Δ3PAL, Δ3PADP, Δ3PADM, and Δ3PAPM respectively. Among them, the CA yields of the strains Δ3PAD, Δ3PAP, and Δ3PADP were not significantly affected, the endotoxin level of the strain Δ3PAP was significantly reduced, and the endotoxin levels of the other two strains were increased instead; the yield of the strain Δ3PAM reached the same level as the original strain at 48 h, and the endotoxin level was also significantly reduced.

[0189] On the basis of the strains Δ3PADP and Δ3PADM, msbA was overexpressed by P12 / P18 / P20 low-medium-high three different strength promoters, and the results showed that msbA overexpression could significantly improve the growth performance of the strains.

[0190] The strain Δ3PAP is selected as the chassis strain for further modification. The 18th amino acid P (proline) or the 50th amino acid P in the msbA gene in the genome is mutated to S (serine) by CRISPR editing technology to obtain two strains named Δ3PAP18 and Δ3PAP50, respectively. The endotoxin level of the two strains is significantly reduced, and the yield level is also affected to a certain extent, which improves the survival ability of the strain to a certain extent. On the basis of the two strains (Δ3PAP18 and Δ3PAP50), the lpxM gene is further knocked out to obtain strains Δ3PAP18M and Δ3PAP50M. On the basis of the above strains, the gene lpxL is further knocked out to obtain strains Δ3PAP18ML and Δ3PAP50ML. The yield of the two strains is further improved compared with strains Δ3PAP18 and Δ3PAP50, the growth state of the strain is better, and the endotoxin level is further reduced.

[0191] According to an embodiment of the present disclosure, the recombinant engineering strain for producing carprofen includes

[0192] a) the lipopolysaccharide core polysaccharide synthesis gene cluster waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG and waaQ on the genome is knocked out or down-regulated; meanwhile, the Lon protein coding gene lon and the HNS regulatory protein coding gene hns are knocked out or down-regulated;

[0193] b) overexpression of Vitreoscilla hemoglobin VHb;

[0194] c) the lpxP gene is knocked out or down-regulated;

[0195] d) the deptA gene is knocked out or down-regulated;

[0196] e) the pagP gene is knocked out or down-regulated;

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

[0198] According to an embodiment of the present disclosure, the recombinant engineering strain for producing carprofen includes

[0199] a) the lipopolysaccharide core polysaccharide synthesis gene cluster waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG and waaQ on the genome is knocked out or down-regulated; meanwhile, the Lon protein coding gene lon and the HNS regulatory protein coding gene hns are knocked out or down-regulated;

[0200] b) overexpression of Vitreoscilla hemoglobin VHb;

[0201] c) the lpxP gene is knocked out or down-regulated;

[0202] d) the deptA gene is knocked out or down-regulated;

[0203] e) the lpxM gene is knocked out or down-regulated;

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

[0205] According to embodiments of the present disclosure, the recombinant engineered bacteria for producing colanic acid comprise

[0206] a) the lipopolysaccharide core polysaccharide synthesis gene cluster waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG and waaQ on the genome are knocked out or down-regulated; meanwhile, the Lon protein encoding gene lon and the HNS regulatory protein encoding gene hns are knocked out or down-regulated;

[0207] b) the V. translucent hemoglobin VHb is overexpressed;

[0208] c) the lpxP gene is knocked out or down-regulated;

[0209] d) the deptA gene is knocked out or down-regulated;

[0210] e) the pagP gene is knocked out or down-regulated;

[0211] f) the lpxM gene is knocked out or down-regulated;

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

[0213] According to embodiments of the present disclosure, the recombinant engineered bacteria further comprise that the lpxM gene is knocked out or down-regulated.

[0214] According to embodiments of the present disclosure, the recombinant engineered bacteria further comprise that the lpxL gene is knocked out or down-regulated.

[0215] According to embodiments of the present disclosure, the recombinant engineered bacteria further comprise that the lpxM gene and the lpxL gene are knocked out or down-regulated.

[0216] According to embodiments of the present disclosure, the recombinant engineered bacteria for producing colanic acid comprise

[0217] a) the lipopolysaccharide core polysaccharide synthesis gene cluster waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG and waaQ on the genome are knocked out or down-regulated; meanwhile, the Lon protein encoding gene lon and the HNS regulatory protein encoding gene hns are knocked out or down-regulated;

[0218] b) overexpression of Vitreoscilla hemoglobin VHb;

[0219] c) lpxP gene is knocked out or down-regulated;

[0220] d) deptA gene is knocked out or down-regulated;

[0221] e) pagP gene is knocked out or down-regulated;

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

[0223] g) lpxM gene and / or lpxL gene is knocked out or down-regulated.

[0224] According to embodiments of the present disclosure, the recombinant engineered bacteria for producing carboxylic acid include

[0225] a) the lipopolysaccharide core polysaccharide synthesis gene cluster waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG and waaQ on the genome is knocked out or down-regulated; meanwhile, the Lon protein encoding gene lon and the HNS regulatory protein encoding gene hns are knocked out or down-regulated;

[0226] b) overexpression of Vitreoscilla hemoglobin VHb;

[0227] c) lpxP gene is knocked out or down-regulated;

[0228] d) deptA gene is knocked out or down-regulated;

[0229] e) lpxM gene is knocked out or down-regulated;

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

[0231] g) lpxM gene and / or lpxL gene is knocked out or down-regulated.

[0232] According to embodiments of the present disclosure, the recombinant engineered bacteria for producing carboxylic acid include

[0233] a) the lipopolysaccharide core polysaccharide synthesis gene cluster waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG and waaQ on the genome is knocked out or down-regulated; meanwhile, the Lon protein encoding gene lon and the HNS regulatory protein encoding gene hns are knocked out or down-regulated;

[0234] b) overexpression of Vitreoscilla hemoglobin VHb;

[0235] c) lpxP gene is knocked out or down-regulated;

[0236] d) the eptA gene is knocked out or down-regulated;

[0237] e) the pagP gene is knocked out or down-regulated;

[0238] f) the lpxM gene is knocked out or down-regulated;

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

[0240] h) the lpxM gene and / or the lpxL gene is knocked out or down-regulated.

[0241] According to an embodiment of the present disclosure, the Vitreoscilla hemoglobin VHb gene comprises

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

[0243] 2) a polynucleotide encoding a variant of SEQ ID NO: 1, which is a sequence based on the amino acid point mutation, deletion and / or addition of SEQ ID NO: 1 and which has 90% or more identity with the sequence of SEQ NO: 1; and / or

[0244] 3) a polynucleotide encoding a sequence from other species with 90% or more identity with SEQ ID NO: 1.

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

[0246] In another aspect of the present disclosure, there is provided use of the recombinant engineering bacteria as previously described in the preparation of a product producing colanic acid.

[0247] In one aspect of the present disclosure, there is provided a method for constructing recombinant engineering bacteria producing colanic acid, comprising:

[0248] a) knocking out or down-regulating the lipopolysaccharide core polysaccharide synthesis gene cluster waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG and waaQ on the genome; and knocking out or down-regulating the Lon protein encoding gene lon and the HNS regulatory protein encoding gene hns;

[0249] b) overexpressing Vitreoscilla hemoglobin VHb;

[0250] c) knocking out or down-regulating the lpxP gene.

[0251] According to embodiments of the present disclosure, the method of constructing a recombinant engineered bacterium for producing colanic acid further comprises knocking out or down a kdsD gene or an eptA gene.

[0252] According to embodiments of the present disclosure, the method of constructing a recombinant engineered bacterium for producing colanic acid further comprises knocking out or down a pagP gene and / or a lpxM gene.

[0253] According to embodiments of the present disclosure, the method of constructing a recombinant engineered bacterium for producing colanic acid further comprises

[0254] i) knocking out or down an eptA gene and a pagP gene; or

[0255] ii) knocking out or down an eptA gene and a lpxM gene; or

[0256] iii) an eptA gene, a pagP gene and a lpxM gene are knocked out or down.

[0257] According to embodiments of the present disclosure, the method of constructing a recombinant engineered bacterium for producing colanic acid further comprises

[0258] a single point mutation modification of msbAP18S or msbAP50S.

[0259] According to embodiments of the present disclosure, the method of constructing a recombinant engineered bacterium for producing colanic acid further comprises knocking out or down a lpxM gene.

[0260] According to embodiments of the present disclosure, the method of constructing a recombinant engineered bacterium for producing colanic acid further comprises knocking out or down a lpxL gene.

[0261] According to embodiments of the present disclosure, the Vitreoscilla hemoglobin VHb gene in the method of constructing a recombinant engineered bacterium for producing colanic acid comprises

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

[0263] 2) a polynucleotide encoding a variant of SEQ ID NO: 1, which is a sequence based on amino acid point mutations, deletions and / or additions of SEQ ID NO: 1 and which has 90% or more identity to the sequence of SEQ NO: 1; and / or

[0264] 3) a polynucleotide encoding a sequence from other species which has 90% or more identity to SEQ ID NO: 1.

[0265] According to the embodiments of the present disclosure, the recombinant engineering bacteria for producing caerulomycin are selected from one or more of the Enterobacteriaceae, 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 is to be construed as an admission that the embodiments described in this disclosure are not entitled to antedate such disclosure by virtue of prior application.

[0267] Also provided are embodiments in which any of the embodiments herein can be combined with any one or more of the other embodiments, unless otherwise stated, and provided that the combinations do not result in inconsistencies or contradictions.

[0268] The technical solutions provided by the present disclosure are described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present disclosure.

[0269] Materials and methods

[0270] Seed medium:

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

[0272] Fermentation medium:

[0273] Yeast powder 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 Sunway), plasmid extraction kit (Shanghai Sunway), DNA marker (Takara)

[0276] Seed culture method:

[0277] Use an inoculation loop to pick a streak on a flat plate lamp, inoculate a single colony in 3-7 mL of seed culture medium, and incubate at 34-38°C for 10-18 h.

[0278] Shaking flask fermentation test culture method:

[0279] The seed liquid was inoculated into the fermentation medium at 1%-10% inoculation amount, and the inoculated fermentation medium was cultured at 25-32°C for 24-48h.

[0280] Polysaccharide detection method:

[0281] (1) Sample treatment:

[0282] After 48h of culture, the fermentation liquid was centrifuged at 10,000-15,000rpm for 20min in a refrigerated centrifuge, and the supernatant was collected; 1.5-4 times of absolute ethanol was added to the supernatant, and the mixture was placed in a refrigerator at 2-8°C overnight. The alcohol-sedimented solution was centrifuged at high speed for 20min, and the supernatant was removed and the precipitate was reserved; an appropriate amount of sterile water was added to dissolve the precipitate, and the obtained solution was the extracted polysaccharide extract.

[0283] (2) CA yield detection by carbazole method:

[0284] Colanic acid (CA) is an acidic polysaccharide composed of D-glucose, L-fucose, D-galactose and D-glucuronic acid. The polysaccharide is hydrolyzed with a sodium tetraborate-containing sulfuric acid solution at high temperature, the hydrolysis product D-glucuronic acid acid reacts with carbazole reagent to generate a purple red compound, and the color intensity of the compound is linearly related to its content within a certain range. The content of CA can be calculated by colorimetry of the glucuronic acid content, thereby obtaining the CA content.

[0285] Example

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

[0287] Lipopolysaccharide synthesis consumes precursors that compete with the CA synthesis pathway, and the genes (clusters) in this pathway are considered for knockout. Genes 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), waaG (gene ID: 948149)), 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)) are knocked out. The genome genes are knocked out using the CRISPR-Cas9 method.

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

[0289] To knock out the relevant genes, the corresponding 1000bp nucleotide sequences upstream and downstream of the genes (clusters) are first amplified by PCR, and are named waaF-arm1, waaF-arm2; LG-arm1, LG-arm2; LQ-arm1, LQ-arm2. The two corresponding upstream and downstream homologous arms are connected into a fragment waaF, LG-arm, LQ-arm using fusion PCR. Then, to construct sgRNA, the sgRNA sequence is designed online using the chopchop website with the corresponding gene sequence as the template, and the obtained sequence is connected to the pTarget plasmid to obtain a plasmid containing the sgRNA of the corresponding gene, which are pTarget-waaF, pTarget-LG, and pTarget-LQ, respectively.

[0290] 2. Preparation of E. coli competent cells

[0291] For gene knockout, the constructed homologous arm fragments and sgRNA plasmid need to be introduced into cells, for which the cells need to be made competent to absorb exogenous DNA. (1) The strain containing the pCas9 plasmid is inoculated into 50 ml of LB medium at an inoculation amount of 1% to 2%, and incubated at 30°C. When the OD600 reaches 0.1 to 0.2, 2 mL of L-arabinose with a concentration of 1 M (final concentration 40 mM) is added, and the culture is incubated at 30°C to induce the expression of the recombinase. The induction time is at least 1 h, and when the OD600 reaches 0.6-0.7, the competent cells are prepared. (2) The bacteria are collected by centrifugation at 4°C and 5000 rpm for 5 min, and the bacterial cells are resuspended by adding pre-cooled sterile 10% glycerol and washing 3 times (washing with 1 mL of glycerol solution). (3) Finally, 500 uL of pre-cooled 10% glycerol is added (100 times concentration of 50 mL of bacterial solution) to prepare competent cells, and 90 uL per tube is aliquoted and stored at -80°C for standby.

[0292] 3. Electroporation knockout

[0293] The obtained homologous arm fragments waaF-arm, LG-arm, LQ-arm and sgRNA pTarget-waaF, pTarget-LG, pTarget-LQ plasmids are added to the competent cells in the order of 1:1 to 1:4 ratio, gently mixed, and ice-bathed for 30 min; then transferred to an electroporation cup for electroporation (1 mm electroporation cup, 1800 V, 200 Ω), and immediately after electroporation, 600 uL of pre-cooled LB medium is added and transferred to a 1.5 mL EP tube, and incubated at 30°C for 2.5-3 h. Then, it is plated on a plate containing spectinomycin and kanamycin, and after incubation at 30°C, the colonies are grown and the knockout results are verified by PCR.

[0294] 4. Elimination of plasmid

[0295] The successfully selected strains are inoculated into 2 mL of LB solution (corresponding to the addition of antibiotic Kan), and a final concentration of 0.5 mM IPTG is added, and incubated at 30°C for 12 h to eliminate the pTarget plasmid. A small amount of bacterial solution is dipped and streaked on a Kan plate, and incubated at 30°C. The single colonies are picked and spotted on Spc and Kan plates. The strain that grows on the Kan plate but not on the Spc plate is the one that has eliminated the pTarget plasmid. The single colonies are inoculated into LB medium and incubated at 37°C (or 42°C) overnight (high temperature causes the loss of the temperature-sensitive plasmid pCas), and a small amount of bacterial solution is dipped and streaked on an antibiotic-free plate and incubated at 37°C. The single colonies are spotted on Kan and antibiotic-free plates and incubated at 37°C. The colonies that grow on the antibiotic-free plate but not on the Kan plate are the strains that have successfully eliminated the plasmid. Finally, the strains ΔF, ΔLG, and ΔLQ with the relevant genes knocked out are obtained.

[0296] The strains ΔF, ΔLG, and ΔLQ obtained by the above knockout were inoculated in seed culture medium for culture, and then transferred to fermentation medium for fermentation test. After high-speed centrifugation of the obtained fermentation liquor, the fermentation liquor supernatant was collected, and the yield of the corresponding three strains was detected by colorimetry to be 0.3 g / L, 0.15 g / L, and 0.4 g / L (Figure 1). The results show that knocking out the genes related to the lipopolysaccharide pathway has a significant promoting effect on the synthesis of CA, and the highest yield is obtained when the gene cluster LQ is knocked out.

[0297] Example 2 Knockout of lon and hns genes

[0298] The same technical means as in Example 1 were used to construct the homologous arms hns-arm and lon-arm and sgRNA plasmids pTarget-hns and pTarget-lon for knocking out hns (gene ID: 945829) and lon (gene ID: 945085) respectively on the basis of the ΔLQ strain. The same method was used for the steps of preparing competent cells, electrotransformation knockout, and plasmid loss to sequentially knock out the lon and hns genes, and obtain the recombinant strain Δ2. The strain was analyzed and verified in a shake flask and a 5-L fermentation tank, and the yield reached 2.2 g / L and 14.5 g / L, respectively, as shown in Figure 2, which was a substantial increase compared with the yield of the control strain.

[0299] Example 3 Overexpression of Vitreoscilla hemoglobin VHb

[0300] The hemoglobin VHb-encoding gene vgb was connected to four different promoters pET, Pvgb, Ptac, and aP4 (the promoter sequences are SEQ ID NO: 2-SEQ ID NO: 5) respectively, and the obtained recombinant plasmids were transformed into the Δ2 strain to obtain recombinant strains named Δ2 / pET-vgb, Δ2 / Pvgb-vgb, Δ2 / Ptac-vgb, and Δ2 / aP4-vgb. The yield of the four strains was analyzed at the shake flask level and the 5-L fermentation tank level, and the results are shown in Figures 3 and 4. The results show that the strain Δ2 / Ptac-vgb (renamed as Δ3) has the highest yield at the shake flask and fermentation tank levels, and the CA yield in the 5-L fermentation tank reaches 16.8 g / L, which is increased by 15.9% compared with the yield of the control.

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

[0302] Based on the above Δ3 strain, the key genes lpxL, lpxP and lpxM in the endotoxin synthesis pathway were knocked out respectively, and only lpxP was successfully knocked out after multiple attempts, and the strain was named Δ3ΔlpxP; Based on the strain Δ3, the knockout of the other two genes was lethal, and the corresponding mutant strains could not be obtained. The yield analysis was carried out in a 5-L fermenter, and the CA yield of the Δ3ΔlpxP strain was 15.4 g / L, and the growth OD600 was even higher than that of the control strain (Figure 5a); The endotoxin level was reduced by 10% compared with the control (Figure 5b). The results show that knocking out the gene lpxP does not affect the growth of the strain, and plays a certain role in reducing endotoxin, but single gene knockout has very limited effect on reducing endotoxin.

[0303] Example 5 Double gene knockout of endotoxin synthesis pathway

[0304] Based on the Δ3ΔlpxP strain, the genes kdsD, eptA, lpxM, and lpxL were further knocked out respectively, and the corresponding recombinant strains were named Δ3PD, Δ3PA, Δ3PM, and Δ3PL. Among them, strains Δ3PM and Δ3PL could not be successfully constructed, and knocking out lpxM or lpxL in the Δ3ΔlpxP strain made the strain lethal. As shown in Figure 6, the performance of Δ3PD and Δ3PA two strains was analyzed and compared in a 5-L fermenter, and the CA yield of the two strains was 14.6 g / L and 14.2 g / L respectively, which was slightly lower than that of the control strain (16.8 g / L); The endotoxin level of the strain was detected, and the endotoxin levels of Δ3PD and Δ3PA were 85.0% and 70.0% respectively relative to the control, and the endotoxin level was further effectively reduced.

[0305] Example 6 Construction of three gene and / or four gene combination modified strains

[0306] Based on the Δ3PA strain, the genes kdsD, pagP, lpxM, lpxL, kdsD+pagP, kdsD+lpxM were further knocked out respectively, and the corresponding recombinant strains were named Δ3PAD, Δ3PAP, Δ3PAM, Δ3PAL, Δ3PADP, Δ3PADM. The results are shown in Figure 7a, the growth state of strains Δ3PAD, Δ3PAP, Δ3PADP is good, and there is little difference with the control, and even the growth state is better than the control strain, such as Δ3PADP; As shown in Figure 7b, the corresponding CA yield is not significantly affected, and the 24h yield is 15.3 g / L, 16.6 g / L and 14.6 g / L respectively. The growth state of strains Δ3PAM and Δ3PADM is obviously slower than that of the original strain, and the CA yield is affected to a certain extent. Exceptionally, the growth of strain Δ3PAM is slow, and the 24h yield is reduced, but the 48h yield still reaches the same level as the original strain, which is 16.8 g / L.

[0307] From the analysis of endotoxin level, only the endotoxin level of strain Δ3PAP and Δ3PAM among the various combination knockout strains constructed appeared a significant decrease, which were 9.5% and 42.5% of the control strain, i.e. the endotoxin level of strain Δ3PAP and Δ3PAM decreased by 90.5% and 57.5% respectively compared with the control. While the endotoxin level of other recombinant strains constructed by other knockout methods all appeared different degrees of increase (as shown in Figure 7c).

[0308] Strain Δ3PAL could not be successfully constructed, and knockout of gene lpxL led to strain lethality.

[0309] Example 7 lpxM gene knockout strain analysis

[0310] The endotoxin of the above-mentioned constructed strains Δ3PAP and Δ3PAM all appeared a significant decrease, and the engineering strategies of the two strains were further superimposed, i.e. strain Δ3PAP was used as the starting strain to further knockout lpxM gene to obtain recombinant strain Δ3PAPM. It was found that the growth performance of the strain was general when cultured under the same conditions as the control strain Δ3, and the final strain OD600 was low, but as shown in Figure 9a. From the analysis of endotoxin level, the endotoxin level of Δ3PAPM constructed appeared a significant decrease, which was reduced by 99.0% compared with the control strain (Figure 9b).

[0311] Example 8 Transport protein msbA overexpression

[0312] In the above-mentioned study, the endotoxin level of strain Δ3PAPM appeared a significant decrease, but the growth of the strain was obviously affected. Further, how to ensure low endotoxin while ensuring normal growth of the strain.

[0313] The above-mentioned fast-growing strain Δ3PADP and slow-growing strain Δ3PADM were selected to overexpress msbA protein with P12 / P18 / P20 low, medium and high strength promoters, and the influence of different expression intensity of the gene on the endotoxin level and CA yield level of the strain was investigated. As shown in Figure 8a, the results showed that overexpression of msbA of a certain intensity could significantly improve the growth performance of the strain (such as strain Δ3PADM / P18-msbA), while overexpression of msbA of other intensity could not necessarily improve the growth performance of the strain; as shown in Figure 8b, the CA yield level appeared different degrees of increase (such as strain Δ3PADM / P20-msbA) or decrease (such as strain Δ3PADP / P12-msbA); the endotoxin level relative to the control all appeared different degrees of increase (Figure 8c). Through overexpression of msbA of different intensity, a strain with low endotoxin level and high CA yield level could not be obtained.

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

[0315] From the analysis of Example 6 and 7, it is found that strains Δ3PAP and Δ3PAPM can significantly reduce the endotoxin level, but the growth rate of strain Δ3PAPM becomes very slow. In order to make the subsequent strain grow normally and further modify the strain, the growth ability of the strain needs to be strengthened. The strain Δ3PAP is selected as the chassis strain to continue the next modification. The CRISPR technology (operation same as Example 8) is used to further modify the strain Δ3PAP. The msbA gene in the genome is mutated from P (proline) at the 18th amino acid or P at the 50th amino acid to S (serine) respectively, and two strains are obtained, which are named Δ3PAP18 and Δ3PAP50 respectively. As shown in Figure 9, the 5-L fermenter verification analysis shows that the growth state of the two strains Δ3PAP18 and Δ3PAP50 returns to the same level as the control strain, and the endotoxin level is significantly reduced compared with the control strain, which is only 10% of the control strain. The corresponding yields are 14.01 g / L and 11.4 g / L respectively.

[0316] Example 1) The 18th amino acid P (proline) or the 50th amino acid P of the msbA gene in the genome is mutated to S (serine) respectively, and two strains are obtained, which are named Δ3PAP18 and Δ3PAP50 respectively. As shown in Figure 9, the 5-L fermenter verification analysis shows that the growth state of the two strains Δ3PAP18 and Δ3PAP50 returns to the same level as the control strain, and the endotoxin level is significantly reduced compared with the control strain, which is only 10% of the control strain. The corresponding yields are 14.01 g / L and 11.4 g / L respectively.

[0317] Example 10 lpxM gene knockout

[0318] From the previous research, it is found that the knockout of lpxM has a positive effect on the reduction of endotoxin, but the knockout of the gene will significantly affect the activity of the strain and the yield of CA (shown in Figure 9b). The strains Δ3PAP18 and Δ3PAP50 obtained by single-point mutation of msbA improve the survival ability of the strain to a certain extent. On the basis of these two strains, the lpxM gene is further knocked out to obtain strains Δ3PAP18M and Δ3PAP50M. The two strains are verified and analyzed in a 5-L fermenter, and the growth state of the two strains is good, and the corresponding yields are 12.4 g / L and 15.9 g / L respectively (Figure 10).

[0319] Example 11 lpxL gene knockout

[0320] In the previous research, directly knocking out the lpxL gene of the strain leads to the death of the strain. Here, based on strains Δ3PAP18M and Δ3PAP50M, the lpxL gene is further knocked out, and the strain does not die. Strains Δ3PAP18ML and Δ3PAP50ML are successfully obtained. As shown in Figure 11, the two strains Δ3PAP18ML and Δ3PAP50ML are verified and analyzed in a 5-L fermenter, and the growth state of the two strains is good, even better than the corresponding strains Δ3PAP18M and Δ3PAP50M. The corresponding yields are 18.1 g / L and 18.3 g / L respectively. The yield of these two strains is further improved compared with before knocking out the lpxL gene.

[0321] Example 12 endotoxin level analysis and comparison

[0322] The endotoxin levels of the above constructed recombinant strains Δ3PAP18M, Δ3PAP50M, Δ3PAP18ML, Δ3PAP50ML and the control strain were compared and analyzed, and the results are shown in Figure 12. The results show that the endotoxin levels of the four strains relative to the control are significantly reduced, among which the endotoxin levels of strains Δ3PAP18ML and Δ3PAP50ML are the lowest, being 18.5 EU / mg and 500 EU / mg, respectively, which are reduced by 99.9% relative to the endotoxin level of the control strain, while the yields of the two strains are shown in Figure 11, reaching 18.1 g / L and 18.3 g / L, respectively. Through a series of engineering strategies, the kanamycin-producing strain that can simultaneously achieve low endotoxin and high yield is finally obtained.

[0323] Table 1 sequence information

[0324] Table 2 primer sequence list

[0325] It should be understood that the application of the present disclosure is not limited to the above examples, and those of ordinary skill in the art can make improvements or changes according to the above description, and all these improvements and changes shall belong to the protection scope of the appended claims of the present disclosure.

[0326] Incorporated by reference

[0327] The entire contents of each patent and scientific document referred to herein are incorporated herein by reference for all purposes.

[0328] Equivalents

[0329] The present application can be embodied in other specific forms without departing from the spirit or essential characteristics thereof. Accordingly, the above described embodiments are to be considered in all respects as illustrative and not restrictive, the scope of the application being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

A recombinant engineered bacterium for producing carlic acid, wherein, The recombinant engineering bacteria comprise a) the knockout or down-regulation of the lipopolysaccharide core polysaccharide synthesis gene cluster waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG and waaQ on the genome; while the Lon protein encoding gene lon and the HNS regulatory protein encoding gene hns are knocked out or down-regulated; b) overexpression of Vitreoscilla hemoglobin VHb; c) the knockout or down-regulation of the lpxP gene. The recombinant engineered bacteria of claim 1, wherein, The recombinant engineering bacteria further comprise the knockout or down-regulation of the kdsD gene or the eptA gene. The recombinant engineering bacteria as claimed in claim 2, wherein, The recombinant engineering bacteria further comprise the knockout or down-regulation of the pagP gene and / or the lpxM gene. The recombinant engineered bacteria of claim 1, wherein, The recombinant engineering bacteria further comprise i) the knockout or down-regulation of the eptA gene and the pagP gene; or ii) the knockout or down-regulation of the eptA gene and the lpxM gene; or iii) the knockout or down-regulation of the eptA gene, the pagP gene and the lpxM gene. The recombinant engineering bacteria as claimed in claim 4, wherein, The recombinant engineering bacteria further comprise i) the single-point mutation modification of msbAP18S or msbAP50S; The recombinant engineering bacteria as claimed in claim 5, wherein, The recombinant engineering bacteria further comprise the knockout or down-regulation of the lpxM gene. The recombinant engineering bacteria as claimed in claim 6, wherein, The recombinant engineering bacteria further comprise the knockout or down-regulation of the lpxL gene. The recombinant engineered bacteria of claim 1, wherein, The Vitreoscilla hemoglobin VHb gene comprises 1) a polynucleotide encoding SEQ ID NO: 1; 2) a polynucleotide encoding a variant of SEQ ID NO: 1, which is a sequence based on the amino acid point mutation, deletion and / or addition of SEQ ID NO: 1 and has more than 90% identity with the sequence of SEQ ID NO: 1; and / or 3) a polynucleotide encoding a sequence from other species with more than 90% identity with SEQ ID NO:

1. The recombinant engineered bacteria of claim 1, wherein, The recombinant engineering bacteria are selected from one or more of the Enterobacteriaceae family, preferably Escherichia coli BL21 (DE3), JM109, Nissle 1917 (EcN), BW23110, or MG1655. The use of the recombinant engineering bacteria as claimed in any one of claims 1-9 in the preparation of a product for producing colanic acid. A method for constructing a recombinant engineering bacteria for producing colanic acid, comprising: a) the knockout or down-regulation of the lipopolysaccharide core polysaccharide synthesis gene cluster waaL, waaU, waaZ, waaY, waaR, waaO, waaB, waaP, waaG and waaQ on the genome; while the Lon protein encoding gene lon and the HNS regulatory protein encoding gene hns are knocked out or down-regulated; b) overexpression of Vitreoscilla hemoglobin VHb; c) the knockout or down-regulation of the lpxP gene. The method of claim 11, wherein, Further comprising the knockout or down-regulation of the kdsD gene or the eptA gene. The method of claim 12, wherein, Further comprising the knockout or down-regulation of the pagP gene and / or the lpxM gene. The method of claim 11, wherein, Further comprising i) the knockout or down-regulation of the eptA gene and the pagP gene; or ii) the knockout or down-regulation of the eptA gene and the lpxM gene; or iii) the knockout or down-regulation of the eptA gene, the pagP gene and the lpxM gene. The method of claim 14, wherein, Further comprising Single-point mutation modification of msbAP18S or msbAP50S. The method of claim 15, wherein, Further comprising knocking out or down lpxM gene. The method of claim 16, wherein, Further comprising knocking out or down lpxL gene. The method of claim 11, wherein, The transparent Vitreoscilla hemoglobin VHb gene comprises 1) a polynucleotide encoding SEQ ID NO: 1; 2) a polynucleotide encoding a variant of SEQ ID NO: 1, which is a sequence based on amino acid point mutation, deletion and / or addition of SEQ ID NO: 1 and which has more than 90% identity with SEQ NO: 1 sequence; and / or 3) a polynucleotide encoding a sequence from other species which has more than 90% identity with SEQ ID NO:

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

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