A bacterium producing pyranosyl glucopyranose lipid A or its derivatives

Abstract

The present invention discloses a bacterium for producing pyranose glucopyranose lipid A or its derivatives, wherein the bacterium is a Gram-negative bacterium, in which a protein for adding a secondary C12 acyl chain at the 2' position of the lipid A structure during the synthesis of lipid A is replaced by a protein for adding a secondary C14 hydroxy acyl chain at the 2' position of the lipid A structure. The present invention synthesizes pyranose glucopyranose lipid A or its derivatives in microorganisms for the first time by modifying Gram-negative bacteria, the overall steps are simple, the cost is greatly reduced compared to the chemical synthesis of GLA, and the invention has good application prospects.

Description

Technical Field

[0001] The present disclosure relates to the field of biotechnology, and in particular, to a bacterium producing glucopyranosyl lipid A (GLA) or a derivative thereof, and a method for preparing glucopyranosyl lipid A or a derivative thereof. Background Art

[0002] Glucopyranosyl lipid A (GLA) is a new generation of TLR4 agonists based on rational design and chemical synthesis. It has 6 acyl side chains, all of which are tetradecanoyl chains. It has been shown to have adjuvant activity superior to MPLA derived from Salmonella minnesota R595, which has been used in a variety of marketed vaccines. At present, GLA has broad application prospects in the fields of vaccine development and tumor immunotherapy. Clinical stage candidate vaccines as adjuvants include tuberculosis, malaria, influenza, HIV, visceral leishmaniasis, schistosomiasis, etc.

[0003] GLA is a non-natural compound and is currently obtained through chemical synthesis. Due to the complex structure of GLA, its chemical synthesis is difficult, with more than 30 steps, complex precursors, limited raw material sources, low yield, and high cost, which is far from meeting market demand. Therefore, it is necessary to find a new method to solve the above problems. Summary of the invention

[0004] Technical issues solved:

[0005] One aspect of the present disclosure is to provide a new bacterium producing pyranosyl glucopyranosyl lipid A or its derivatives in order to address the problems in the prior art of complex preparation methods, low yield and high cost of pyranosyl glucopyranosyl lipid A or its derivatives.

[0006] Specifically, in order to reduce the acquisition cost of pyranose glucopyranose lipid A or its derivatives, the present invention uses a synthetic biology method to obtain the target product through strain modification and then strain fermentation to adapt to large-scale production and solve the above technical problems.

[0007] Technical solutions provided:

[0008] A bacterium producing pyranose glucopyranosyl lipid A or its derivatives, the bacterium being a Gram-negative bacterium, in which a protein for adding a secondary C12 acyl chain at the 2' position of the lipid A structure during lipid A synthesis is replaced by a protein for adding a secondary C14 hydroxy acyl chain at the 2' position of the lipid A structure.

[0009] In some embodiments of the present disclosure, the purpose of the present disclosure is achieved by modifying the genome of the bacterium. In the genome of the bacterium, the gene encoding the protein that adds a secondary C12 acyl chain at the 2' position of the lipid A structure is replaced by the gene of the protein that adds a secondary C14 hydroxyl acyl chain at the 2' position of the lipid A structure.

[0010] In some embodiments of the present disclosure, the Gram-negative bacteria is Escherichia coli, which belongs to the genus Escherichia.

[0011] In Escherichia coli, in order to add a secondary 14-carbon hydroxy acyl chain to the 2' position of the lipid A structure, the secondary C12 acyl chain (12C→14C) at the original 2' position is replaced to construct a lipid A product with 6 C14 acyl chains. In some embodiments of the present disclosure, in the genome of the bacterium, the lpxL gene can be replaced by the vplpxL gene (VP_RS00880 or VP_RS08405), the cslpxL gene (YP_001438378.1) or the kplpxL gene (WP_008805482.1). In one embodiment of the present disclosure, in the genome of the bacterium, the lpxL gene is replaced by the vplpxL gene (VP_RS00880). The NCBI accession numbers of the sequences of the above-mentioned lpxL gene, vplpxL gene (VP_RS00880), vplpxL gene (VP_RS08405), cslpxL gene and kplpxL gene are 946216, 1187646, 1189252, YP_001438378.1 and 69735137, respectively.

[0012] In Escherichia coli, in order to reduce the consumption of precursors for lipid A synthesis by competitive pathways, or to reduce possible by-products, or to reduce unnecessary modifications to lipid A, so as to better achieve the purpose of the present disclosure, in some embodiments of the present disclosure, in the genome of the bacteria, the function of one or more genes selected from the following is deleted or reduced: lipid A palmitoyl modification gene, phosphoethanolamine modification gene, phosphate modification gene or 4-amino-4-deoxy-L-arabinose modification gene. Preferably, in some embodiments of the present disclosure, the lipid A palmitoyl modification gene includes lpxP gene and / or pagP gene, the phosphoethanolamine modification gene includes eptA gene and / or eptB gene, the phosphate modification gene includes lpxT gene, and the 4-amino-4-deoxy-L-arabinose modification gene includes arnT gene. The NCBI accession numbers of the sequences of the above-mentioned lpxP gene, pagP gene, eptA gene, eptB gene, arnT gene and lpxT gene are 946847, 946360, 948629, 948068, 947297 and 946693, respectively.

[0013] In one embodiment of the present disclosure, the Escherichia coli strain obtained after transformation is represented as △lpxP, △pagP, △eptA, △eptB, △lpxT, △arnT, lpxL::vplpxL and is named WL strain.

[0014] Furthermore, in E. coli, in order to hydrolyze the phosphate at position 1 of the lipid A molecule and relieve the inhibitory effect of regulatory proteins on the expression of exogenous genes inserted into the lacZ site later, so as to form constitutive expression, thereby better achieving the purpose of the present disclosure, in some embodiments of the present disclosure, the lacI gene is knocked out on the chromosome of the bacteria, and the hplpxE gene encoding 1-phosphotransferase from Helicobacter pylori is knocked in. The NCBI accession number of the above-mentioned hplpxE gene sequence is 66521281.

[0015] In one embodiment of the present disclosure, the Escherichia coli strain obtained after further transformation is represented as △lpxP, △pagP, △eptA, △eptB, △lpxT, △arnT, lpxL::vplpxL, △lacI-lacZ::hplpxE, and is named WLE strain.

[0016] The LPS produced by the WL strain and the WLE strain obtained above has the same acyl chain structure as GLA. After further hydrolysis and purification, pyranose glucopyranosyl lipid A or its derivatives can be obtained.

[0017] Further, in Escherichia coli, in order to obtain an engineered strain that can directly produce pyranose glucopyranose lipid A or its derivatives, in some embodiments of the present disclosure, the function of the gene encoding heptose epimerase (rfaD) in the genome of the bacteria is missing or reduced. Heptose epimerase (rfaD) is the last step reaction enzyme in the ADP-heptose synthesis pathway, a precursor of LPS core polysaccharide synthesis. The functional loss or reduction of the gene encoding this protein blocks the synthesis of the core polysaccharide precursor. The above-mentioned rfaD gene has an NCBI accession number of 948134.

[0018] Further, in Escherichia coli, in order to obtain a target product without core polysaccharide, in some embodiments of the present disclosure, the function of the gene (waaA) encoding 3-deoxy-D-mannose-octanoyltransferase (Kdo) in the genome of the bacteria is lost or reduced. A method for achieving the loss of waaA gene function, for example, a base C148T mutation occurs in the chromosomal lipopolysaccharide transporter gene msbA (the 50th proline encoded by it mutates to serine), and the msbA148 mutant protein has the ability to flip lipid A out of the inner membrane, alleviate the Kdo-dependent growth of the mutant strain, and give it the ability to survive in the absence of the Kdo sugar biosynthesis pathway, thereby further knocking out waaA. The NCBI accession number of the above-mentioned msbA gene is 945530, and the NCBI accession number of the waaA gene is 949048.

[0019] In one embodiment of the present disclosure, the Escherichia coli strain obtained after further transformation is represented by △rfaD, △lpxP, △pagP, △eptA, △eptB, △lpxT, △arnT, lpxL::vplpxL, △lacI-lacZ::hplpxE, msbA::msbA148, △waaA, and is named W2E△A strain.

[0020] The W2E△A strain obtained above can synthesize GLA from scratch without acid hydrolysis, and the extraction process is simpler.

[0021] Another aspect of the present disclosure is to provide use of the above-mentioned bacteria in preparing pyranosyl glucopyranose lipid A or its derivatives.

[0022] Another aspect of the present disclosure is to provide a method for preparing pyranose glucopyranosyl lipid A or a derivative thereof, comprising:

[0023] 1) amplifying the above bacteria, collecting the obtained bacteria and extracting lipopolysaccharide solution, hydrolyzing the obtained lipopolysaccharide, and purifying the obtained hydrolyzate to obtain the pyranosyl glucopyranose lipid A or its derivative; or

[0024] 2) amplifying the above bacteria, collecting the obtained bacteria and performing lipid extraction, and purifying the obtained total lipid extract to obtain the pyranose glucopyranosyl lipid A or its derivatives.

[0025] Another aspect of the present disclosure is to provide a bacterial composition, which includes at least two of the above-mentioned bacteria. The included bacteria can be mixed in any proportion. The bacterial composition described in the present disclosure can achieve the same or better technical effects as the above-mentioned bacteria.

[0026] Another aspect of the present disclosure provides a method for preparing a vaccine adjuvant, comprising the step of preparing pyranose glucopyranosyl lipid A or its derivatives using the above-mentioned bacteria or the above-mentioned composition.

[0027] Beneficial effects:

[0028] The present invention synthesizes pyranose glucopyranose lipid A or its derivatives in microorganisms for the first time by modifying Gram-negative bacteria. The overall steps are simple and the cost is greatly reduced compared with the chemical synthesis of GLA, which has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the PCR verification result of the WL strain;

[0030] Figure 2 This is the PCR verification result of the WLE strain;

[0031] Figure 3 This is the PCR verification result of W2E△A strain;

[0032] Figure 4 This is a TLC test result diagram of the fermentation product;

[0033] Figure 5A and Figure 5B This is the result of the target product time-of-flight mass spectrometer (MALDI-TOF) analysis, where Figure 5A In the positive ion mode, Figure 5B It is negative ion mode;

[0034] Figure 6 This is the LC-MS-MS result diagram of the fermentation product and the standard;

[0035] Figure 7 This is a diagram showing the activity test results of the product. DETAILED DESCRIPTION

[0036] The present invention discloses a bacterium that produces pyranose glucopyranosyl lipid A (GLA) or its derivatives. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention, and relevant personnel can obviously modify or appropriately change and combine the contents described herein without departing from the content, spirit and scope of the present invention to achieve and apply the technology of the present invention.

[0037] In the present invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise expressly stated, throughout the specification and claims, the term "include" or its transformations such as "include" or "include" etc. will be understood to include the elements or components stated, without excluding other elements or other components. The term "one (a)" ("a", "an" and "the") includes plural indicators. The term "multiple (multiple)" refers to two (kinds) or more (kinds). The terms "such as", "for example" etc. are intended to refer to exemplary embodiments, and are not intended to limit the scope of the present disclosure.

[0038] In the present disclosure, when a range of values ​​is provided, it should be understood that the endpoints are included in the range and each intervening value between the upper and lower limits of the range and any other specified value or intervening value in the specified range and any value in the smaller range between the specified values ​​are included unless the context clearly dictates otherwise.

[0039] In this disclosure, the term "about" generally refers to variations within a range of 0.5%-10% above or below a specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0040] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. The definition of common terms in molecular biology can be found in Lewin's GENES, Twelfth Edition, Jocelyn E. Krebs, Elliott S. Goldstein, Stephen T. Kilpatrick, Publisher: Jones & Bartlett Learning. The definition of common terms in biochemistry can be found in Lehninger Principles of Biochemistry, Eighth Edition, David L. Nelson, Michael M. Cox, Publisher: WH Freeman. The definition of common terms in cell biology can be found in Molecular Biology of the Cell, Sixth Edition, Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter, Publisher: Garland Science. The definition of common terms in genetics can be found in Genetics: Analysis of Genes and Genomes, Eighth Edition, Daniel L. Hartl, Maryellen Ruvolo, Publisher: Jones & Bartlett Learning.

[0041] Unless otherwise specified, the experimental techniques herein employ conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which can be found in standard books such as the following: Molecular Cloning: A Laboratory Manual; Cell Biology: A Laboratory Handbook, etc.

[0042] the term:

[0043] The term "glycopyranoside lipid A" (GLA), chemical formula C 96 H 181 N2O 22P, with a molecular mass of 1745.28, has a structure including 2 aminoglucose sugars, 1 phosphate group and 6 fourteen-carbon fatty acid chains (with a β-(1'-6)-linked D-glucosamine disaccharide as the backbone, phosphorylated at the 4' position, the 3' and 2' amino groups connected to (R)-3-(tetradecyloxy)tetradecyl, respectively, and the 3 and 2 amino groups connected to tetradecyloxy, respectively), and the specific structural formula is shown in Formula I.

[0044]

[0045] GLA can be used as a vaccine adjuvant. GLA adjuvant can stimulate and induce innate and adaptive immune responses by inducing maturation of dendritic cells (DC) and the release of related proinflammatory cytokines and chemokines. Studies have confirmed that TLR4 agonist pyranosyl lipid A (GLA-SE) as an adjuvant for H5N1 influenza vaccine has good immune effects in Phase II clinical trials and has been tested in tuberculosis vaccines and tumor antigen vaccines.

[0046] The term "derivative" may also be referred to as a "variant", which refers to a compound molecule obtained by subjecting the original compound molecule to any form of modification or change. In some embodiments of the present disclosure, the derivative of the pyranose glucopyranosyl lipid A may include a compound molecule obtained by replacing atoms or atomic groups in its structure, such as fatty acid chains, glucose groups, etc. In some embodiments of the present disclosure, the derivative is derived from a product obtained by not knocking out, for example, lipid A palmitoyl modification genes, phosphoethanolamine modification genes, phosphate modification genes, or 4-amino-4-deoxy-L-arabinose modification genes in the genome of the bacterium. These products retain the basic structure and basic function of pyranose glucopyranosyl lipid A.

[0047] The term "gram-negative bacteria" refers to fungi that appear red in a gram staining experiment. Gram staining is a method for distinguishing bacteria by differences in bacterial cell wall structure. In some embodiments of the present disclosure, the gram-negative bacteria include, for example, Escherichia coli, Shigella dysenteriae, Proteus, Klebsiella pneumoniae, Bordetella pertussis, Shigella, Yersinia, Salmonella, Haemophilus influenzae, Bacillus cholerae, Bacillus parahaemolyticus, Acinetobacter, Haemophilus parainfluenzae, Bordetella parapertussis, and strains of the above genera or species. In the present disclosure, the gram-negative bacteria have the common feature of releasing lipopolysaccharide (LPS).

[0048] The term "lipid A" is an important component of the outer membrane of the cell wall of Gram-negative bacteria, which is mainly composed of lipopolysaccharide (LPS), including three parts: polysaccharide side chains (O antigens), core polysaccharides and lipid A. Lipid A is the main toxic component of endotoxins, which can anchor in the outer leaflet of the outer membrane and bind to Toll-like receptors to trigger an immune response. Lipid A has important functions in medicine. It is one of the main pathogenic factors of Gram-negative bacterial infections, and can recognize TLR4 on the surface of host immune cells, trigger a series of signal transductions, and ultimately synthesize cytokines such as IL-8 and TNF-α. In addition, lipid A can also bind to LBP in serum, catalyze lipopolysaccharide binding to CD14, stimulate inflammatory response, lead to a decrease in immune defense function, and may cause serious health problems. Studies have shown that the structural diversity of lipid A is closely related to its pathogenicity. For example, the lipid A structure of Francisella cannot be recognized by TLR4 of the host immune system, which is related to its virulence.

[0049] The term "replacement" in the present disclosure means, for example, that compared with the original parent bacteria, one protein is replaced by another protein, and one gene is replaced by another gene, so that the function of the replaced protein or gene is lost, and then changed to the function of the new protein or gene. Its methods are known in the prior art. In one embodiment of the present disclosure, gene editing technology is used to achieve site-specific replacement of the target gene or protein, and the gene editing method is, for example, the CRISPR / Cas9 system.

[0050] The term "Escherichia coli" (scientific name: Escherichia coli), also known as Escherichia coli. It is a species of the genus Escherichia in the family Enterobacteriaceae. Escherichia coli is a Gram-negative, facultative anaerobic, rod-shaped, Escherichia coli bacterium with blunt ends, motile, non-spore-forming, and warm-blooded organisms commonly found in the lower part of the small intestine. This bacterium can be easily and cheaply grown and cultured in a laboratory environment and is widely used as a host for gene replication and expression in biological experiments. Escherichia coli is also a chemotrophic type, and its chemically defined culture medium must contain a carbon source and an energy source. Escherichia coli is the most widely studied prokaryotic model organism and an important species in the fields of biotechnology and microbiology. It is a host organism in most recombinant DNA work. Under favorable conditions, replication only takes 20 minutes. Common Escherichia coli include, for example, BL21 (DE3) strain, BL21 (DE3) pLysS strain, W3110 strain, DH5α strain, JM109 strain, HB101 strain, TOP10 strain, etc. In one embodiment of the present disclosure, the Escherichia coli is strain W3110.

[0051] The term "loss of gene function" refers to a state in which a specific gene loses its normal function in an organism through a certain technical means. The above functions may include encoding proteins, transcriptional regulation, and regulating epigenetic modifications of the genome. The loss of target gene function can be achieved using techniques known in the prior art. For example, in some embodiments of the present disclosure, a gene mutation method is used to introduce point mutations, insertions, or deletions to cause the target gene to lose its normal function. In other embodiments of the present disclosure, a gene knockout technique is used to cause the target gene to lose its normal function. "Gene knockout" refers to the directed destruction of a gene in a cell or body, resulting in a complete loss of its function. In some embodiments of the present disclosure, the knockout of a target gene can be achieved by a variety of methods in the prior art. For example, rendering a target gene non-functional by inserting an unrelated gene into the target gene, such as by homologous recombination, or by any other method that disrupts gene function in cells; RNAi methods, which use small RNA molecules to silence or degrade the messenger RNA (mRNA) of the target gene, preventing it from being translated into protein; gene editing methods based on the CRISPR system, such as the CRISPR / Cas9 system, which uses guide RNA (sgRNA) and the Cas9 enzyme to introduce targeted DNA breaks at specific locations in the genome, resulting in gene disruption.

[0052] The term "reduced gene function" refers to reducing the expression level of a gene in an organism by specific technical means, rather than completely eliminating the function of the gene. In some embodiments of the present disclosure, reducing the function of the target gene may lead to a reduction in the yield and purity of the preparation process of GLA or its derivatives, but it does not affect the purpose of the present invention. The reduction of gene function can be achieved by means of gene knockdown. In some embodiments of the present disclosure, it can be achieved by methods such as interference (CRISPRi) of antisense RNA, ribonuclease or CRISPR-Cas system. In the present disclosure, the reduction of the function of the gene is compared with the function of the original gene, and is reduced by at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 97%, about 99%.

[0053] The term "constitutive expression" refers to continuous expression in almost all cells of an individual organism. The expression of such genes is less affected by environmental factors, but is continuously expressed or changes very little in most or almost all tissues of the individual at all growth stages. Constitutive expression is in contrast to inducible expression. Inducible expression refers to certain genes that are not expressed or expressed at a very low level under normal circumstances, but under the action of inducers (such as metabolites), the expression of the gene is activated or enhanced. Constitutive expression has no spatiotemporal specificity, that is, it is not affected by period, location, or environment.

[0054] Bacterial culture:

[0055] In the present disclosure, the fermentation culture method of bacteria can be used in any stage of the bacterial preparation process of the present invention.

[0056] The culture medium used in the culture may be LB medium (Luria-Bertani medium), beef extract peptone medium, starch agar medium, potato medium, etc. The culture medium may or may not include antibiotics.

[0057] The bacterial culture method may be, for example, the method described in Fermentation Engineering Experiment, edited by Li Jianghua, 2011.

[0058] Hydrolysis of LPS:

[0059] In some embodiments of the present disclosure, LPS prepared by bacteria has the same acyl chain structure as GLA, which is hydrolyzed to obtain the pyranose glucopyranosyl lipid A or its derivatives. The hydrolysis of lipopolysaccharide usually needs to be carried out under certain conditions, including temperature, pH value, enzymes or chemical reagents, etc. Different hydrolysis conditions will affect the rate of hydrolysis and the type of products. For example, the use of specific enzymes (such as polysaccharide hydrolases) can effectively hydrolyze the polysaccharide chain part of lipopolysaccharide under milder conditions. There are various methods for hydrolyzing lipopolysaccharides, including chemical hydrolysis, enzymatic hydrolysis, and a comprehensive hydrolysis method combining these two methods. In one embodiment of the present disclosure, acid is used for hydrolysis. In an acidic solution, the molecules of lipopolysaccharide will be partially degraded. The acid can be hydrochloric acid, sulfuric acid, acetic acid, etc.

[0060] Extraction of lipid products:

[0061] In the present disclosure, the lipid product can be extracted using a suitable method in the prior art. For example, in some embodiments of the present disclosure, the methods that can be used are: chloroform-methanol method, hydrolysis method, ultrasonic method, extraction method (the extractant is an organic solvent, such as chloroform, n-hexane, petroleum ether, etc.), enzymatic method, gas chromatography, high performance liquid chromatography (HPLC), phenol-chloroform method, evaporation concentration method, freezing method, etc.

[0062] Purification of the product:

[0063] In the present disclosure, any suitable method in the prior art can be used to purify the product pyranose glucopyranosyl lipid A or its derivatives. For example, in some embodiments of the present disclosure, the methods that can be used include: precipitation method, extraction method, chromatography method, centrifugation method, chromatography method, membrane separation method, etc.

[0064] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with specific embodiments.

[0065] Example:

[0066] Example 1: Construction of WL strain and WLE strain

[0067] The lpxP, pagP, eptA, eptB, arnT and lpxT genes in the chromosome of E. coli W3110 were inactivated by deletion mutation; then, the lpxL gene was knocked out and replaced with the vplpxL gene from Vibrio parahaemolyticus to obtain the WL strain; on this basis, the hplpxE gene from Helicobacter pylori was knocked in at the lacI-lacZ site to obtain the WLE strain.

[0068] The method mainly comprises the following steps: firstly, in order to reduce possible by-products, lipid A palmitoyl modification genes (lpxP, pagP), phosphoethanolamine modification genes (eptA, eptB), phosphate modification gene (lpxT) and 4-amino-4-deoxy-L-arabinose (L-Ara4N) modification gene (arnT) are sequentially knocked out in the chromosome of Escherichia coli W3110 strain to obtain an intermediate strain W1; then, the acyl chain length of lipid A is modified, and the acyl modification gene lpxL is replaced with Vibrio parahaemolyticus (Vibrio parahaemolyticus) parahaemolyticus) acyltransferase vplpxL gene, the protein encoded by which can add a secondary 14-carbon hydroxy acyl chain at the 2' position of lipid A structure, replacing the original secondary C12 acyl chain (12C→14C) at the 2' position, constructing a lipid A product with 6 C14 acyl chains, and obtaining the WL strain, with the genotype of W3110△lpxP,△pagP,△eptA,△eptB,△lpxT,△arnT,lpxL::vplpxL. The gene verification results are as follows Figure 1 As shown, lanes 1-6 correspond to the band sizes after knocking out the lpxP, pagP, eptA, eptB, lpxT and arnT genes, respectively, and lane 7 is the band size after knocking in vplpxL.

[0069] Then, based on the WL strain, the 1-phosphotransferase hplpxE gene from Helicobacter pylori was knocked into the chromosome lacI-lacZ site. The protease it encodes can hydrolyze the phosphate at the 1 position of lipid A molecules; the lacI gene encodes the regulatory protein in the lac operon. Knocking out the lacI gene can release the inhibitory effect of the regulatory protein on the expression of the exogenous gene inserted into the lacZ site later, so that it forms constitutive expression, and the WLE strain is obtained. The genotype is W3110△lpxP,△pagP,△eptA,△eptB,△lpxT,△arnT,lpxL::vplpxL,△lacI-lacZ::hplpxE. The gene verification results are as follows Figure 2As shown, lanes 1-6 correspond to the band sizes after knocking out lpxP, pagP, eptA, eptB, lpxT and arnT genes, respectively, lane 7 is the band size after knocking in vplpxL, and lanes 8 and 9 are the band sizes at both ends after knocking in hplpxE at the lacI-lacZ position.

[0070] The LPS produced by the WL and WLE strains has the same acyl chain structure as GLA.

[0071] The genes involved above include lpxP, pagP, eptA, eptB, arnT and lpxT gene knockout fragments, and the NCBI accession numbers of their sequences are "946847", "946360", "948629", "948068", "947297" and "946693", respectively.

[0072] The NCBI accession numbers of the sequences of the lpxL gene fragment and the vplpxL gene replacement fragment are 946216 and 1187646 respectively.

[0073] The lacI and lacZ gene knockout fragments and the hplpxE gene insertion fragment have sequences with NCBI accession numbers of 945007, 945006 and 66521281 respectively.

[0074] The intermediate strains or final strains constructed above were verified by gene sequencing.

[0075] Example 2: Construction of W2EΔA strain

[0076] The rfaD, lpxP, pagP, eptA, eptB, arnT and lpxT genes in the E. coli W3110 chromosome were inactivated by deletion mutation, the lpxL gene was replaced with the vplpxL gene from Vibrio parahaemolyticus, the hplpxE gene from Helicobacter pylori was knocked into the lacI-lacZ site, the msbA gene in the chromosomal genome underwent base C148T (i.e., residue P50S), and the waaA gene underwent a deletion mutation.

[0077] The method mainly comprises the following steps: firstly, knocking out the heptose epimerase (rfaD) in the chromosome of the Escherichia coli W3110 strain, which is the last step reaction enzyme in the synthesis pathway of ADP-heptose, a precursor of LPS core polysaccharide synthesis, thereby blocking the synthesis of the core polysaccharide precursor and constructing the intermediate strain W0; secondly, successively knocking out the palmitoyl transferase genes (lpxP, pagP), phosphoethanolamine transferase genes (eptA, eptB), phosphotransferase genes (lpxT) and 4-amino-4-deoxy-L-arabinose transferase genes (arnT) of lipid A, reducing the related modifications to the lipid A molecules, thereby reducing possible byproducts and constructing the intermediate strain W1; thirdly, the acyl transferase gene lpxL is replaced by the vplpxL gene from Vibrio parahaemolyticus, the protein encoded by which can add a fourteen carbon hydroxy fatty acid chain at the 2' position of the lipid A structure, replacing the original The secondary C12 acyl chain at the 2' position (12C→14C) was removed to obtain a lipid A product with 6 C14 acyl chains, and the intermediate strain W2 was constructed. Then, based on the W2 strain, the hplpxE gene (lpxE is derived from Helicobacter pylori, and the protease it encodes can hydrolyze the phosphate at the 1 position of the lipid A molecule) was knocked in at the lacI-lacZ site of the chromosome. The lacI gene encodes a regulatory protein in the lac operon. Knocking out this gene can release the inhibitory effect of the regulatory protein on the expression of the exogenous gene inserted into the lacZ site later, so that it forms constitutive expression, and the intermediate strain W2E is constructed. Finally, in order to knock out the 3-deoxy-D-mannose-octanoate (Kdo) transferase gene (waaA) and obtain the target product without core polysaccharide, the base C148T mutation (the 50th proline encoded by it is mutated to serine) occurred in the chromosomal lipopolysaccharide transporter gene msbA on the chromosome. The msbA148 mutant protein has the function of converting lipid The ability of A to flip out of the inner membrane alleviates the Kdo-dependent growth of the mutant strain and gives it the ability to survive in the absence of the Kdo sugar biosynthesis pathway, thereby further knocking out waaA and constructing the W2E△A strain. The genotype is W3110△rfaD,△lpxP,△pagP,△eptA,△eptB,△lpxT,△arnT,lpxL::vplpxL,△lacI-lacZ::hplpxE,msbA::msbA148,△waaA.

[0078] Genetic verification results Figure 3As shown, lanes 1-7 correspond to the band sizes after knocking out rfaD, lpxP, pagP, eptA, eptB, lpxT and arnT genes, respectively, lane 8 is the band size after knocking in vplpxL, lane 9 is the band size after knocking in hplpxE, lane 10 is the band size of amplified msbA, and lane 11 is the band size after knocking out waaA.

[0079] The W2E△A strain can synthesize GLA from scratch without the need for acid hydrolysis, making the extraction process simpler.

[0080] The NCBI accession number of the above gene fragment rfaD is 948134, the NCBI accession number of msbA is 945530, and the NCBI accession number of waaA is 949048.

[0081] The intermediate strains or final strains constructed above were verified by gene sequencing.

[0082] Example 3: Obtaining GLA

[0083] 1. Fermentation culture of the obtained strain

[0084] The cultivation and fermentation of the genetically engineered bacteria are as follows:

[0085] 1) Culture medium preparation: LB culture medium was used as seed culture medium, and the ingredients were: 10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast extract. Fermentation medium: 24 g / L yeast extract, 12 g / L peptone, 3 g / L potassium dihydrogen phosphate, 6.78 g / L disodium hydrogen phosphate, 0.5 g / L sodium chloride, 1 g / L ammonium chloride, 5 g / L glucose, 0.5 g / L magnesium sulfate, 0.5 ml / L defoamer. Feed medium: 600 g / L glycerol, 8 g / L magnesium sulfate, 0.2 ml / L defoamer. After preparation, sterilize at 115°C for 30 min, and the glucose and magnesium sulfate in the fermentation medium are sterilized separately or filtered.

[0086] 2) Primary seed solution culture: a single colony of the W7 strain was placed in a test tube containing 3.5 mL of seed culture medium and cultured overnight at 37° C. and 200 rpm to obtain a primary seed solution.

[0087] 3) Secondary seed solution culture: All the primary seed solution was inoculated into a 2 L shake flask containing 350 mL of seed culture medium, and cultured at 37°C and 200 rpm for 8 h to obtain the secondary seed solution.

[0088] 4) Fermentation tank culture: All the secondary seed liquid was inoculated into a 15L fermentation tank containing 7L fermentation medium, the temperature was 37°C, pH was maintained at 7.0 with ammonia water and 20% phosphoric acid solution, the speed was set to be associated with dissolved oxygen, dissolved oxygen was 30%, and the speed was 250-800rpm. After 6 hours of culture, feeding was performed at a rate of 1mL / min, and fermentation was terminated after 24 hours of culture to obtain a fermentation liquid.

[0089] 2. Extraction and acid hydrolysis of WL and WLE lipopolysaccharides

[0090] Strains WL and WLE contain a complete LPS structure, and obtaining the target product GLA from them is divided into two steps: extraction of lipopolysaccharide and acid hydrolysis of lipopolysaccharide.

[0091] The extraction methods of lipopolysaccharide for both are the same, and the specific steps are as follows: 1) After bacterial fermentation is completed, the strains are collected by centrifugation and washed twice;

[0092] 2) Resuspend the cells in 70%-90% ethanol solution, sonicate in a 60°C water bath for 30 minutes, centrifuge at 3000 g for 10 minutes, and discard the supernatant;

[0093] 3) Add preheated hot water and phenol mixed liquid (ratio 1:1, water: 90% phenol, v / v), add the mixed liquid according to the final wet bacteria concentration of 20% (w / v), stir at 65℃-70℃ for 30 minutes, and cool to room temperature;

[0094] 4) Centrifuge at 4°C, take the aqueous phase, dialyze, and freeze-dry to obtain lipopolysaccharide.

[0095] The hydrolysis process of lipopolysaccharide is as follows:

[0096] 1) Adjust the lipopolysaccharide concentration to 10-20 mg / ml with pure water, heat under reflux to 95°C, and then add hydrochloric acid (applicable to WL and WLE strains) or acetic acid (applicable to WLE strains);

[0097] 2) Stir and reflux at 95°C for 30 minutes;

[0098] 3) Cool to about 40°C, add 5 times the volume (LPS volume) of chloroform:methanol (2:1, v / v) solution, mix thoroughly and add to a separatory funnel, and take out the lower organic phase after separation;

[0099] 4) Remove the solvent by rotary evaporation to obtain an acid hydrolysis sample.

[0100] 3. Extraction of WLEΔA strain products

[0101] This strain no longer has a complete structure of LPS and can directly produce GLA. The extraction method is as follows:

[0102] 1) Collection and washing of bacterial solution: centrifuge at 3000 rpm for 15 min to collect bacterial solution; resuspend the bacteria in an equal volume of 0.1 M sodium chloride solution and then centrifuge;

[0103] 2) Resuspension of the cells: The volume of the cells is V. Use 1.6 times the volume (1.6V) of 1M sodium chloride to resuspend the cells.

[0104] 3) Lysis of bacterial solution: add 2V chloroform and 4V methanol, at which time the mixture has chloroform:methanol:1MNaCl=1:2:0.8 (v / v / v); mix thoroughly.

[0105] 4) Extraction: add 2V chloroform, 2V volume 1M sodium chloride, adjust chloroform: methanol: 1M NaCl to 2:2:1.8 (v / v / v), mix thoroughly, let stand and take the lower organic phase;

[0106] 5) The organic phase was rotary evaporated to obtain a crude lipid extract.

[0107] Example 4: Purification of product

[0108] The product obtained in Example 3 was purified using an anion exchange medium or an affinity medium, and the two processes were similar, as follows:

[0109] 1) Column packing and column washing

[0110] a. The column was filled with 30 ml column volume corresponding to 10 L fermentation product, and the inner diameter of the selected chromatography column was 26 mm.

[0111] b. Wash the column with 5 times the volume of filler material, 5 times the volume of 0.1N NaOH, 5 times the volume of pure water, 2 times the volume of methanol, and 2 times the volume of chloroform:methanol (4:1, v / v) in sequence.

[0112] 2) Solvent and Filtration of Products

[0113] a. Dissolve the sample in chloroform:methanol (4:1, v / v) solution;

[0114] b. 0.45 μm PVDF membrane filtration.

[0115] 3) Sample loading and elution

[0116] a. Slowly load the sample after filtration;

[0117] b. Wash with 5 volumes of chloroform:methanol (4:1, v / v), and then wash with 5 volumes of chloroform:methanol:water (2:3:1, v / v / v);

[0118] 4) Elution of adsorbed substances

[0119] a. Wash with 5 volumes of chloroform:methanol:ammonium acetate (2:3:1, v / v / v) respectively, where the concentrations of ammonium acetate are 10mM, 20mM, 40mM, 80mM, 160mM, 320mM, and 480mM;

[0120] b. Collect the effluents of different concentrations, add 1 / 2 volume of chloroform and 0.2N HCl, mix thoroughly and separate the layers, take the lower layer of extract, analyze by TLC, combine the components containing the target product, and rotary evaporate.

[0121] 5) Product acidification

[0122] a. Spin dry the sample, add 100 ml of chloroform: methanol: HCl (0.2N) to resuspend, let stand for 30 min, add 100 ml of chloroform, separate the layers, take the chloroform layer, and rotary evaporate to obtain the acidified product.

[0123] Example 5: Identification of products

[0124] The purified product obtained in Example 4 was identified.

[0125] 1. Thin layer chromatography analysis

[0126] Use a capillary to spot the sample on the TLC plate and let it dry. Then use chloroform: methanol: water: ammonia (40:25:4:2, v / v / v / v) to develop it. After the chromatography is completed, take out the plate and blow it dry. The plate is then developed with iodine and 10% sulfuric acid ethanol solution. The results are as follows Figure 4 As shown in the results, it can be seen that most of the impurities can be removed through preliminary purification, and a product with a simpler composition and one phosphate group removed can be obtained.

[0127] 2. MALDI-TOF analysis

[0128] The purified samples were subjected to time-of-flight mass spectrometry analysis with a detection range less than m / z 2500. Figure 5A As shown, the molecular weight of GLA is 1745.3, and the m / z value is 1768.3 in the positive ion mode, which is consistent with Figure 5AThe main peaks of the product obtained in Example 3 are consistent, which proves that the product obtained in Example 3 is GLA. In addition, since some acyl side chains are easily lost during the extraction or purification of lipid A, there are peaks with m / z values ​​of 1558.1, 1331.8 and 1121.6 in the extraction of the strains constructed in Examples 1 and 2. Among these peaks, the peak at m / z = 1558.1 is GLA with five acyl side chains produced by losing a secondary acyl chain; the peak at m / z = 1331.8 is GLA with four acyl side chains produced by losing a secondary C14 acyl chain and a primary C14 acyl chain; the peak at m / z = 1121.6 is GLA with three acyl side chains produced by losing two secondary C14 acyl chains and a primary C14 acyl chain.

[0129] Negative ion detection mode was used with a detection range less than m / z 2500. Figure 5B As shown in Figure 2, the molecular weight of GLA is 1745.3, and the m / z value in negative ion mode is 1744.3, which is consistent with Figure 5B The main peaks in the figure are consistent. The peak at m / z=1534.1 is the GLA with five acyl side chains produced by the loss of a secondary acyl chain; the peak at m / z=1307.9 is the GLA with four acyl side chains produced by the loss of a secondary C14 acyl chain and a primary C14 acyl chain; the peak at m / z=1097.7 is the GLA with three acyl side chains produced by the loss of two secondary C14 acyl chains and one primary C14 acyl chain.

[0130] 3. LC-MS analysis

[0131] Chemically synthesized commercial GLA (PHAD) was used as a standard for LC-MS analysis. Figure 6 As shown, the results show that the target product has the same retention time and molecular weight as GLA, proving that GLA was obtained by biosynthesis in this example.

[0132] Example 6: Activity determination of products

[0133] Activity assay was performed using HEK-Blue-hTLR4 cell line, the process is as follows:

[0134] (1) Cell plating: 2×10 cells per well 5 96-well plate;

[0135] (2) Sample addition: Sodium chloride was used as a negative control, LPS was used as a positive control, 10 ng per well, and the GLA obtained by the present invention was set at three different concentrations, 1 ng, 5 ng, and 10 ng per well;

[0136] (3) Detection: After adding the color developer, start timing and measure the absorbance at a wavelength of 650 nm every hour for a total of 12 hours;

[0137] (4) Processing results: Figure 7 As shown, GLA has a good effect in stimulating hTLR4. A significant difference can be seen after 4 hours of stimulation, and the advantage becomes more obvious afterwards. Even the effect of 1 ng added is not inferior to that of 10 ng LPS.

[0138] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A bacterium producing pyranosyl glucopyranose lipid A, characterized in that: The bacteria is Escherichia coli, Wherein, in the genome of the bacterium, the lpxL gene is replaced by the vplpxL gene, and the vplpxL gene is a gene encoding an acyltransferase derived from Vibrio parahaemolyticus; In the genome of the bacterium, the functions of the following genes are deleted or reduced: lpxP gene, pagP gene, eptA gene, eptB gene, lpxT gene and arnT gene.

2. The bacterium according to claim 1, characterized in that The hplpxE gene is knocked into the chromosome of the bacterium, wherein the hplpxE gene is a gene encoding 1-phosphotransferase derived from Helicobacter pylori.

3. The bacterium according to claim 1 or 2, characterized in that The function of the gene encoding heptose epimerase in the genome of the bacterium is deleted or reduced.

4. The bacterium according to claim 3, characterized in that The function of the gene encoding 3-deoxy-D-mannose-octulosyltransferase in the genome of the bacterium is deleted or reduced.

5. Use of the bacterium as claimed in any one of claims 1 to 4 in the preparation of pyranose glucopyranosyl lipid A.

6. A method for preparing pyranose glucopyranosyl lipid A, characterized in that: include: 1) amplifying the bacteria according to claim 1 or 2, collecting the obtained bacteria and extracting lipopolysaccharide solution, hydrolyzing the obtained lipopolysaccharide, and purifying the obtained hydrolyzate to obtain the pyranosyl glucopyranose lipid A; or 2) Amplifying the bacteria described in claim 3 or 4, collecting the obtained bacteria and performing lipid extraction, and purifying the obtained total lipid extract to obtain the pyranose glucopyranosyl lipid A.

7. A bacterial composition, characterized in that The composition comprises at least two of the bacteria as described in any one of claims 1 to 4.

8. A method for preparing a vaccine adjuvant, characterized in that: The method comprises the step of preparing pyranose glucopyranosyl lipid A using the bacterium described in any one of claims 1 to 4 or the composition described in claim 7.

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