CN114438146A - Method for producing histidine by microbial fermentation and application - Google Patents

Abstract

The invention discloses a method for producing histidine by microbial fermentation. The method can produce histidine with higher efficiency and higher yield by mainly increasing the action of ATP phosphoribosyltransferase in microorganisms and blocking purine repressor.

Description

Method for producing histidine by microbial fermentation and application

Technical Field

The invention relates to a high-histidine-yield genetic engineering escherichia coli strain, a construction method thereof and a method for producing histidine by fermenting the strain. The invention also relates to a method for recovering histidine from a fermentation process. Belonging to the fields of medicine, food and feed.

Background

L-Histidine (L-Histidine), generally in the form of the hydrochloride salt, is used industrially. Histidine hydrochloride is colorless crystal or white crystalline powder; almost no odor, slightly salty taste; the aqueous solution shows an acidic reaction. Is easily soluble in water and insoluble in ethanol, diethyl ether or chloroform. The melting point is 249-253 ℃.

The traditional preparation method of L-histidine is to use hemoglobin as a raw material, hydrolyze the hemoglobin with hydrochloric acid, then use ion exchange resin to carry out column chromatography separation, and then use hydrochloric acid to recrystallize. The conventional means is: is extracted from pig blood and cattle blood. Spray drying pig blood to obtain blood powder, hydrolyzing with hydrochloric acid, concentrating the eluate containing L-histidine until crystallization occurs, adjusting pH to 2.5 with hydrochloric acid while hot, immediately adding 2 times of ethanol, standing, precipitating, filtering to obtain crude L-histidine hydrochloride, decolorizing, recrystallizing, and drying to obtain the final product.

In recent years, the production process of L-histidine mainly adopts animal hair or feather protein as a raw material. However, there is a risk of other viruses from animal sources, such as bovine spongiform encephalopathy, avian influenza, etc., and there is an increasing tendency to find products produced using non-animal sources.

There have also been attempts to extract L-histidine from hydrolysates of plant materials such as soybean, but the production of L-histidine from defatted soybean is difficult to achieve in large quantities due to low yield and high cost, and has not yet been commercialized.

Japanese Ajinomoto corporation has disclosed a patent, introduced a microbial fermentation method for the production of L-histidine new method. The inventors of this patent have studied and found that L-histidine can be produced in high yield from a bacterium belonging to the genus Bacillus containing a vector containing a genetic factor associated with histidine antagonist resistance, which is obtained from a chromosome of a mutant strain belonging to the genus Bacillus having histidine antagonist resistance. In addition, in 2016, 1 month, the food safety Committee of Japan, 591 th, food safety Committee, health impact evaluation was performed on the food additive "L-histidine hydrochloride produced by the HIS-No.2 strain", and the health impact evaluation results were summarized (case). HIS-No.2 strain was constructed by introducing E.coli K-12 strain-derived L-histidine synthesis-related gene into Escherichia coli K-12 strain-derived mutant strain, which is a strain derived from Escherichia coli K-12, as a host. On the basis of obtaining high-yield strains, the company utilizes a microbial fermentation technology to commercially produce L-histidine on a large scale, and develops corresponding fermentation and purification technology processes.

According to the invention, according to the principle of microbial metabolic engineering, firstly, the endogenous purine repressor (PurR) of the microorganism is deleted, the purR expression inhibition on purA is relieved, and the ATP vacancy-supplementing synthesis speed is increased. Secondly, an ATP Phosphoribosyl Transferase (HisG) mutant which is insensitive to the negative feedback inhibition of L-histidine is screened and overexpressed to improve the effect of the ATP Phosphoribosyl Transferase in the microorganism, so that the microorganism can produce the L-histidine with higher efficiency and higher yield. And finally, removing mycoprotein, pigment and salt by using a shake flask and fermentation tank fermentation optimization process and membrane filtration processes such as ceramic membrane, ultrafiltration and the like, and then separating and extracting the L-histidine hydrochloride. The novel process not only can ensure the safety of the process and products, but also can provide high-quality L-histidine for the market.

Disclosure of Invention

The invention aims at the metabolic pathway of histidine, adopts a novel genetic modification method to transform microorganisms, and utilizes the microorganisms to produce histidine with higher efficiency and higher yield.

Specifically, the present invention increases L-histidine synthesis by screening gene mutants of ATP phosphoribosyltransferase to release the negative feedback limitation of HisG activity by L-histidine and overexpressing the mutant enzymes. Meanwhile, endogenous purine repressor (PurR) is deleted, purR expression inhibition on purA is relieved, ATP deficiency synthesis speed is increased, and therefore the microorganisms can produce histidine with higher efficiency and higher yield.

According to one embodiment of the present invention, the present invention relates to a method for producing histidine by microbial fermentation, comprising:

A) culturing a microorganism in a fermentation medium, said microorganism comprising at least one genetic modification that increases the action of ATP phosphoribosyltransferase in the microorganism; and

B) collecting the histidine produced from the culturing step A).

In the present invention, the genetic modification that increases the action of ATP phosphoribosyltransferase in a microorganism is selected from the group consisting of a) an increase in the enzymatic activity of ATP phosphoribosyltransferase in a microorganism; and/or b) ATP phosphoribosyltransferase is overexpressed in the microorganism;

it will be appreciated by those skilled in the art that to enhance the action of ATP phosphoribosyltransferase in a microorganism, it can be achieved by screening for mutants encoding genes having increased enzymatic activity of ATP phosphoribosyltransferase. Screening hisG gene mutants can be accomplished by obtaining high frequency mutant genes by error-prone PCR techniques. In order to improve the action of HisG in microorganisms, it can also be achieved by overexpressing HisG by increasing the gene copy number thereof, replacing a promoter having a higher expression level than the native promoter, or the like.

In a specific embodiment, the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that increases the action of ATP phosphoribosyltransferase in the microorganism.

In a preferred embodiment, the microorganism is transformed with at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding an ATP phosphoribosyltransferase.

In one aspect, the nucleic acid sequence encoding ATP phosphoribosyltransferase comprises at least one genetic modification that increases the enzymatic activity of ATP phosphoribosyltransferase; further preferably, the genetic modification comprises a modification in a nucleotide sequence corresponding to the amino acid sequence SEQ ID NO:12 at one or more of the following positions: histidine 232 substituted with leucine, threonine 252 substituted with serine, and glutamic acid 271 substituted with glycine; more preferably, the nucleic acid sequence encoding ATP phosphoribosyltransferase is SEQ ID NO: 11.

in another aspect, the ATP phosphoribosyltransferase has an amino acid sequence that is at least about 30% identical, preferably at least about 50% identical, more preferably at least about 70% identical, more preferably at least about 80% identical, even more preferably at least about 90% identical, and most preferably at least about 95% identical to the amino acid sequence of SEQ ID NO 12, wherein the ATP phosphoribosyltransferase has enzymatic activity; further preferably, the ATP phosphoribosyltransferase has the amino acid sequence of SEQ ID NO 12.

In another aspect, the recombinant nucleic acid molecule has an increased copy number of a gene encoding an ATP phosphoribosyltransferase.

In another aspect, the recombinant nucleic acid molecule comprises an endogenous native promoter or a promoter having a higher expression level than the endogenous native promoter; preferably, the promoter having a higher expression level than the endogenous native promoter is selected from the group consisting of HCE promoter, gap promoter, trc promoter, T7 promoter; further preferably, the promoter having a higher expression level than the endogenous native promoter is a trc promoter.

In another preferred embodiment, the microorganism comprises at least one genetic modification to an endogenous native promoter of a gene encoding ATP phosphoribosyltransferase; preferably, the endogenous native promoter of the gene encoding ATP phosphoribosyltransferase is replaced by a promoter with a higher expression level; further preferably, the promoter having a higher expression level is selected from the group consisting of HCE promoter, gap promoter, trc promoter, T7 promoter; most preferably, the promoter with the higher expression level is the trc promoter.

In the present invention, the recombinant nucleic acid molecule transforms a microorganism selected from the group consisting of episomal (i.e., the recombinant nucleic acid molecule is incorporated into a plasmid) and integrative (i.e., the recombinant nucleic acid molecule is integrated into the genome of the microorganism). Preferably, the recombinant nucleic acid molecule is integrated into the genome of the microorganism.

According to a preferred embodiment of the invention, the microorganism further comprises at least one genetic modification capable of reducing the action of the purine repressor PurR in the microorganism.

In one aspect of the above embodiments, the genetic modification that reduces the action of the purine repressor PurR in a microorganism includes, but is not limited to: a partial or complete deletion, or a partial or complete inactivation, of an endogenous gene encoding the purine repressor PurR in the microorganism and/or a partial or complete deletion, or a partial or complete inactivation, of an endogenous native promoter encoding the purine repressor PurR gene in the microorganism. Preferably, the genetic modification which reduces the action of the purine repressor PurR in the microorganism is a complete deletion, i.e.a deletion, of the endogenous gene coding for the purine repressor PurR in the microorganism. In a particular embodiment, the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification capable of reducing the action of the purine repressor PurR in the microorganism.

In one aspect of any of the above embodiments, the expression of the recombinant nucleic acid molecule is inducible, including but not limited to induction by lactose. For example, lactose-induced expression can be achieved by adding lactose or the like to the culture medium.

It will be appreciated by those skilled in the art that a variety of conventional fermentation media known in the art may be used in the present invention. In one aspect, the fermentation medium comprises a carbon source. In another aspect, the fermentation medium comprises a nitrogen source. In another aspect, the fermentation medium comprises a carbon source and a nitrogen source. In another aspect, the fermentation medium comprises a carbon source, a nitrogen source, and inorganic salts.

It will be appreciated by those skilled in the art that a variety of carbon sources known in the art may be used in the present invention, including organic carbon sources and/or inorganic carbon sources. Preferably, the carbon source is selected from one or more of glucose, fructose, sucrose, galactose, dextrin, glycerol, starch, syrup and molasses. Preferably, the concentration of the carbon source is maintained at about 0.1% to about 5%. It will be appreciated by those skilled in the art that a variety of nitrogen sources known in the art may be used in the present invention, including organic nitrogen sources and/or inorganic nitrogen sources. Preferably, the nitrogen source is selected from one or more of ammonia, ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium acetate, sodium nitrate, urea, yeast extract, meat extract, peptone, fish meal, bean meal, malt, corn steep liquor and cotton seed meal.

Preferably, the present invention employs a fed-batch fermentation process. According to one aspect of the invention, the glucose-supplemented liquid comprises glucose, preferably, at a glucose concentration of 10% to 85% (w/v), more preferably, at a glucose concentration of 55% to 75% (w/v).

In a preferred embodiment, the culturing step is performed at about 20 ℃ to about 45 ℃, and more preferably, the culturing step is performed at about 33 ℃ to about 37 ℃.

In a preferred embodiment, the culturing step is carried out at about pH4.5 to about pH 8.5. Further preferably, the culturing step is carried out at about pH6.7 to pH 7.2.

It will be appreciated by those skilled in the art that histidine can be collected using various conventional methods known in the art in the present invention. Preferably, histidine can be collected from extracellular products in the fermentation medium. Further preferably, the collecting step comprises a step selected from the group consisting of: (a) precipitating histidine from the microbially removed fermentation broth; and/or (b) crystallizing histidine from the microorganism-depleted fermentation broth.

According to the present invention, the collecting step further comprises a step of decolorizing the fermentation broth. The decolorization step can include, but is not limited to, one or more precipitation or crystallization resolubilizations of the fermentation broth prior to precipitation or crystallization of the fermentation broth, including activated carbon treatment and/or chromatographic decolorization. The chromatographic decolorization includes the step of contacting the fermentation broth with an ion exchange resin, including but not limited to an anion exchange resin and/or a cation exchange resin, such as contacting the fermentation broth with a mixed bed of anion and cation exchange resins.

In the present invention, the microorganism may be any microorganism (e.g., bacteria, protists, algae, fungi or other microorganisms). In preferred embodiments, the microorganism includes, but is not limited to, bacteria, yeast, or fungi. Preferably, the microorganism is selected from bacteria or yeast. Further preferably, the bacteria include, but are not limited to, bacteria of a genus selected from the group consisting of Escherichia (Escherichia), Bacillus (Bacillus), Lactobacillus (Lactobacillus), Pseudomonas (Pseudomonas) or Streptomyces (Streptomyces); more preferably, the bacteria include, but are not limited to, bacteria of a species selected from Escherichia coli (Escherichia coli), Bacillus subtilis (Bacillus subtilis), Bacillus licheniformis (Bacillus licheniformis), Lactobacillus brevis (Lactobacillus brevis), Pseudomonas aeruginosa (Pseudomonas aeruginosa) or Streptomyces lividans (Streptomyces lividans). Further preferably, the yeast includes, but is not limited to, a yeast selected from the group consisting of Saccharomyces (Saccharomyces), Schizosaccharomyces (Schizosaccharomyces), Candida (Candida), Hansenula (Hansenula), Pichia (Pichia), Kluyveromyces (Kluveromyces), and Rhodofavus (Phaffia); more preferably, the yeast includes, but is not limited to, a yeast selected from the group consisting of Saccharomyces cerevisiae (Saccharomyces cerevisiae), Schizosaccharomyces pombe (Schizosaccharomyces pombe), Candida albicans (Candida albicans), Hansenula polymorpha (Hansenula polymorpha), Pichia pastoris (Pichia pastoris), Pichia canadensis (Pichia canadensis), Kluyveromyces marxianus (Kluyveromyces marxianus), and Phaffia rhodozyma (Phaffia rhodozyma). Preferably, the microorganism is a fungus; further preferably, the fungus includes, but is not limited to, a fungus of a genus selected from Aspergillus (Aspergillus), Absidia (Absidia), Rhizopus (Rhizopus), Chrysosporium (Chrysosporium), Neurospora (Neurospora) or Trichoderma (Trichoderma); more preferably, the fungus includes, but is not limited to, a fungus selected from Aspergillus niger (Aspergillus niger), Aspergillus nidulans (Aspergillus nidulans), Absidia coerulea (Absidia coerulea), Rhizopus oryzae (Rhizopus oryzae), Rakennocardia Chrysosporium (Chrysosporium lucknowense), Neurospora crassa (Neurospora crassa), Neurospora intermedia (Neurospora intermedia) or Trichoderma reesei (Trichoderma reesei). Particularly preferred E.coli strains include K-12, B and W, with K-12 being most preferred. Although E.coli is the preferred microorganism and is used as an example of various embodiments of the present invention, it is understood that any other microorganism that produces histidine and can be genetically modified to increase histidine production can be used in the methods of the present invention. The microorganisms used in the present invention may also be referred to as production organisms.

In the present invention, the term histidine refers to L-histidine. The term L-histidine can be referred to as (S) -2-amino-3- (4-imidazolyl) propionic acid, L-a-amino-beta-4-imidazolyl propionic acid. The term L-histidine may be abbreviated to L-His, respectively.

The term enhancing the action of an enzyme in a microorganism means that the activity of the enzyme in the microorganism is increased and/or that the enzyme is overexpressed, thereby increasing the amount of product produced by the substrate catalyzed by the enzyme in the microorganism.

The term reducing the action of an enzyme in a microorganism means that the activity of the enzyme in the microorganism is reduced and/or that the expression of the enzyme is reduced, thereby reducing the amount of product produced by a substrate catalysed by the enzyme in the microorganism.

The term increased enzymatic activity refers to an increased ability to catalyze a chemical reaction. It encompasses an increased ability of the enzyme to catalyze a chemical reaction by itself with unchanged enzyme product inhibition and enzyme affinity for the substrate, and/or an increased ability to catalyze a chemical reaction due to decreased enzyme product inhibition and/or increased enzyme affinity for the substrate. The term reduced product inhibition of an enzyme means that the activity of the enzyme catalyzing the reaction is reduced by its end-product specific inhibition. The term increased affinity of an enzyme for a substrate refers to an increased affinity of an enzyme for the substrate being catalyzed.

FIG. 1 illustrates, by way of example, E.coli, the main aspects of the genetic modifications disclosed in the present invention for use in the metabolic pathway for large-scale production of histidine. Fig. 1 discloses a method for the synthesis of histidine, including modifications to HisG. It will be appreciated by those skilled in the art that other microorganisms have similar pathways for sugar metabolism, and that genes and proteins have similar structures and functions in such pathways. Thus, the discussion of the present invention applies equally to other microorganisms than E.coli, and other microorganisms are expressly included in the present invention.

It is known in the art that enzymes having the same biological activity may have different names depending on from what microorganism the enzyme is derived. The following are alternative names for many of the enzymes referred to herein and the names of specific genes encoding such enzymes from some organisms. The names of these enzymes may be used interchangeably or, if appropriate, for a given sequence or organism, but the present invention is intended to include enzymes of a given function from any organism.

For example, an enzyme generally referred to herein as "ATP phosphoribosyltransferase", which refers to ATP-dependent phosphoribosyltransferase, is the second enzyme in the L-histidine synthesis pathway, is a key enzyme in the overall reaction, and is also subject to feedback inhibition by the final product L-histidine. The negative feedback limitation of the L-histidine on the ATP phosphoribosyl transferase activity is relieved, and the synthesis of the L-histidine can be increased. ATP phosphoribosyl transferase (ATP-phosphoribosyl transferase) from Escherichia coli (Escherichia coli) is generally referred to as HisG. ATP phosphoribosyltransferases from various organisms are well known in the art and can be used in the genetic engineering strategy of the present invention. For example, it is described herein that ATP phosphoribosyltransferase from escherichia coli has a sequence defined by SEQ ID NO:7, or a pharmaceutically acceptable salt thereof.

For example, an enzyme, generally referred to herein as a "purine repressor," inhibits transcription of the pur operon by interacting with the DNA of the regulatory region of the operon. And (3) deleting endogenous purine repressor, relieving purR from purA expression inhibition, and improving ATP vacancy-supplementing synthesis speed. The purine repressor protein from E.coli (Escherichia coli) is generally referred to as PurR. For example, it is described herein that the purine repressor protein from e.coli has the sequence defined by SEQ ID NO:4, or a nucleotide sequence represented by seq id no.

The "Trc promoter" is skillfully designed for prokaryotic expression, such as an E.coli expression system. The Trc promoter is well known in the art and can be used in the genetic engineering strategy of the present invention. For example, the Trc promoter described herein has the amino acid sequence of SEQ ID NO: 13, or a nucleotide sequence represented by seq id no.

The invention has the beneficial effects that: the invention proves that the negative feedback limitation of L-histidine on the HisG activity can be relieved by screening the gene mutant of ATP phosphoribosyltransferase, the action of ATP phosphoribosyltransferase in microorganisms is improved by overexpression, and the synthesis of L-histidine is increased. Meanwhile, endogenous purine repressor is deleted, purR inhibition on purA expression is relieved, ATP vacancy-filling synthesis speed is increased, and therefore the microorganism can produce histidine with higher efficiency and higher yield.

Each of the publications and references cited or described herein is incorporated by reference in its entirety.

Drawings

FIG. 1 shows the genetic modification in the histidine biosynthetic and metabolic pathways in E.coli.

Detailed Description

The present invention will be described in further detail with reference to specific examples. The following examples are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of the invention. All techniques implemented based on the teachings of this disclosure are intended to be within the scope of this disclosure.

Unless otherwise indicated, the starting materials and reagents used in the examples are all commercially available products.

Example 1

This example describes genetic modifications to reduce the action of purine repressor in microorganisms by deleting the endogenous purine repressor protein (PurR).

The purine repressor PurR inhibits transcription of the pur operon by interacting with the DNA of the regulatory region of the operon. The gene sequence of endogenous purine repressor PurR is deleted to relieve purR from purA expression inhibition, and ATP deficiency synthesis speed is increased, so that pur operon expression regulation is improved. The parent strain ZAS-001 is an E.coli strain that has been partially genetically manipulated.

1. Preparation of Linear DNA full-Length PCR fragment for Red recombinant targeting

(1) PCR amplification of fKanrf fragments

The fKanrf fragment, namely the FRT-Kanr-FRT fragment, means that base sequences of FRT sites specifically recognized by FLP recombinase are arranged at two ends of a kalamycin resistance gene (Kanr).

Designing a primer: the forward primer (mfKanf-F) SEQ ID NO:1 and the reverse primer (mfKanf-R) SEQ ID NO: 2.

Template: pPic 9K.

And (3) PCR reaction conditions: the first step is as follows: denaturation at 94 deg.C for 1 min; the second step is that: 30s at 94 ℃, 30s at 55 ℃ and 40s at 72 ℃ and circulating for 30 times; the third step: extension at 72 ℃ for 10 min.

fKanrf size: 1.28 kb. The nucleotide sequence of the polypeptide is SEQ ID NO. 3.

The PCR product was separated by 1% agarose gel electrophoresis, purified and the fragment recovered.

(2) PCR amplification of full-length linear DNA fragment for Red recombinant targeting

Designing a homology arm primer: according to the PurR nucleotide sequence SEQ ID NO. 4, a forward primer (PurRKO-F) SEQ ID NO. 5 and a reverse primer (PurRKO-R) SEQ ID NO. 6 of a homology arm for deleting the PurR sequence are designed.

Template: amplified fKanrf PCR fragment.

And (3) PCR reaction conditions: the first step is as follows: denaturation at 94 deg.C for 1 min; the second step is that: 30s at 94 ℃, 30s at 55 ℃ and 40s at 72 ℃ and circulating for 30 times; the third step: extension at 72 ℃ for 10 min.

Amplification products: homology arm + fKanrf + homology arm.

And carrying out agarose gel electrophoresis separation, purification and recovery on the PCR product to obtain a linear DNA full-length PCR fragment of 100 ng/mu l for Red recombinant targeting.

2. Red recombination operation

First, pKD46 vector was transferred into the parent strain E.coli ZAS-001 strain. Then, the prepared targeting linear DNA fragment was electrically transformed, and positive clones were selected. Finally, the resistance gene was eliminated.

(1) Transformation of pKD46 plasmid

The pKD46 carrier is a plasmid with expression Red recombinant enzyme gene, expresses Exo, Bet and Gam three gene segments, 3 genes are arranged under an arabinose promoter, and can be expressed in large quantity by L-arabinose induction. For the purpose of modifying a gene of interest on a chromosome by Red recombination, it is necessary to transform the pKD46 plasmid into escherichia coli.

1) And (3) competent preparation: firstly, the Escherichia coli ZAS-001 strain stored at-20 ℃ is inoculated into 10ml of LB liquid medium at 37 ℃ and 225rpm in a ratio of 1:50-100, and cultured with shaking for 2-3 hours. Adding the culture solution into 10ml centrifuge tube, 4000g × 5min, discarding supernatant, and ice-bath 0.1M CaCl₂5ml were suspended for 5 min. Finally, centrifugation at 4000 g.times.5 min removed the supernatant and washed with ice-bath 0.1M CaCl₂5ml of suspension. Standing at-4 deg.C for 12 hr, and naturally settling. Wherein, 0.1M CaCl₂The preparation of (1): with anhydrous CaCl₂1M CaCl₂Sterilizing with steam pressure of 15lbf/in2 under high pressure for 20min, subpackaging 1.5ml, storing at-20 deg.C, thawing, and diluting at a ratio of 1:10 to obtain 0.1M CaCl₂。

2) And (3) plasmid transformation: mu.l of naturally settled cells were taken and 5. mu.l of pKD46 plasmid was added thereto at-4 ℃ for 30 min. Then, water bath was carried out at 42 ℃ for 1.5min, 0.7ml of SOC medium was added, and shaking was carried out at 30 ℃ for 2 hours. 0.2ml of the bacterial solution was applied to an ampicillin (Amp) plate. Incubated at 30 ℃ overnight (12-16 hours). Selecting single clone, adding into 5ml LB liquid culture medium to culture, extracting plasmid and identifying. And preserving the positive strains for later use.

(2) Electrically converting the prepared linear DNA fragment for targeting, and screening positive clone

1) Preparing electrotransformation competence: the pKD 46-containing E.coli ZAS-001 strain was inoculated into a test tube containing ampicillin LB medium, shaken overnight at 250rpm, inoculated into Amp-containing LB medium in an amount of 1% the next day, cultured at 30 ℃ until OD is reached₆₀₀After reaching about 0.2%, 0.2% L-arabinose was added and the mixture was induced at 30 ℃ for 35 minutes until OD reached₆₀₀Reaching about 0.4. Cooling in ice bath. Washing with ultrapure water once, washing with 10% glycerol twice, and finally resuspending with 10% glycerol in an amount such that the thallus is concentrated 500-fold and 1000-fold to obtain a final concentration.

2) Electric shock conversion: the 2mm electric rotor was taken out from 70% ethanol, washed 2 times with sterilized ultrapure water, and irradiated with an ultraviolet lamp for 30 minutes. Precool for 30 minutes at 4 ℃. And (3) taking 90 mu l of finally resuspended cells, transferring the cells into a precooled centrifuge tube, adding 5 mu l (more than 100 ng) of the full-length PCR fragment (linear DNA) obtained in the step (1), gently sucking and uniformly mixing the cells by using a gun, and carrying out ice bath for 30 minutes. Electric transfer parameters: 2500V, 200. omega., 25. mu.F.

3) Recovery and screening of positive clones: 1ml of LB liquid medium was added thereto at 37 ℃ and 100rpm for 1 hour. One kanamycin (Kan) plate was then coated every 200. mu.l for a total of 5. And (6) uniformly coating and drying. Incubated at 30 ℃ for 24 hours. Clones growing under resistance to the kanamycin were selected for PCR identification and positive clones were selected.

The obtained strains are numbered: ZAS-002(ZAS-001,. DELTA.PurR:: fKanrf).

3. Elimination of resistance genes

For the convenience of subsequent work, the resistance genes in the obtained species (positive clones) were eliminated. The elimination of the resistance gene can be accomplished with the aid of the pCP20 plasmid. pCP20 is a plasmid with ampicillin and chloramphenicol resistance genes, and can express FLP recombinase after heat induction, the enzyme can specifically recognize FRT sites, and the sequence between the FRT sites can be deleted through recombination, and only one FRT site is reserved.

pCP20 was transferred to the above-described kanamycin-resistant clone, cultured at 30 ℃ for 8 hours, then raised to 42 ℃ overnight, and heat-induced FLP recombinase expression was observed, with the plasmid gradually lost. And (3) scratching a plate on an antibiotic-free culture medium by using an inoculating loop dipped bacterial liquid, picking out a grown single clone, and dropping the single clone on a kalamycin resistance plate, wherein the non-grown clone is a clone in which the kalamycin resistance gene is deleted by an FLP recombinase. The identification primers were used as PCR to identify clones with the disappearance of resistance to kanamycin.

The obtained strains are numbered: ZAS-003(ZAS-001,. DELTA.purR).

Example 2

This example describes cloning of the ATP phosphoribosyltransferase gene hisG, screening of gene mutants, transformation of parent strains.

The ATP phosphoribosyltransferase HisG is the first enzyme in the synthetic pathway of L-histidine, is a key enzyme in the whole reaction, and is also subjected to feedback inhibition by the final product L-histidine. The strain is transformed by amplifying the E.coli hisG gene under the control of the Trc promoter to over-express it, the desired increase being L-histidine synthesis. To further increase the amount of histidine synthesis in the production strain, mutants of the gene encoding ATP phosphoribosyltransferase with increased enzymatic activity were selected. The negative feedback limitation of HisG activity by L-histidine is released. To achieve this, the cloned gene is amplified by an error-prone PCR technique, and the gene is amplified by a DNA polymerase for amplification under conditions that result in high frequency mismatches, so as to obtain high frequency mutations in the PCR product.

1. Cloning of escherichia coli HisG gene and preparation of recombinant bacterium

The amino acid sequence of the E.coli hisG gene SEQ ID NO 7 was obtained by searching for NC-007779.1 according to NCBI. Base optimization is carried out according to the base codon preferred by the escherichia coli, and the nucleotide sequence of the nucleotide is SEQ ID NO. 8. The gene of SEQ ID NO 8 was synthesized, and the 5 '-end was ligated with the Nco I cleavage site sequence and the 3' -end with the HindIII cleavage site sequence, which was ligated with pUC57-T vector and sequenced to identify, thereby obtaining the plasmid hisG/pUC 57.

1) Preparing a recombinant plasmid: the plasmid hisG/pUC57 and the vector pTrc99A were digested with NcoI and HindIII, respectively, and the hisG fragment and pTrc99A fragment were separated by agarose gel electrophoresis, purified and recovered, ligated with T4DNA ligase at 16 ℃ overnight, and identified to obtain the recombinant plasmid hisG/pTrc 99A.

2) And (3) competent preparation: first, the frozen ZAS-003 bacteria preserved at-20 deg.C were inoculated into 10ml LB liquid medium at 37 deg.C, 225rpm, at a ratio of 1:50-100, and cultured with shaking for 2-3 hours. Adding the culture solution into 10ml centrifuge tube, 4000g × 5min, discarding supernatant, and ice-bath 0.1M CaCl₂5ml were suspended for 5 min. Finally, centrifugation at 4000 g.times.5 min removed the supernatant and washed with ice-bath 0.1M CaCl₂5ml of suspension. Standing at-4 deg.C for 12 hr, and naturally settling.

3) And (3) plasmid transformation: mu.l of the naturally sedimented cells were taken and added with 5. mu.l of HisG/pTrc99A plasmid at-4 ℃ for 30 min. Then, water bath was carried out at 42 ℃ for 1.5min, 0.7ml of SOC medium was added, and shaking was carried out at 30 ℃ for 2 hours. 0.2ml of the bacterial solution was applied to a penicillin plate. Incubated at 30 ℃ overnight (12-16 hours). Selecting single clone, adding into 5ml LB liquid culture medium to culture, extracting plasmid and identifying. And preserving the positive strains for later use. The recombinant strain transformed from hisG/pTrc99A to ZAS-003 was obtained and named: ZAS-004(ZAS-001, hisG/pTrc99, Δ purR).

2. Error-prone PCR amplification of Bifidobacterium breve ATP phosphoribosyltransferase gene HisG

The method comprises the steps of utilizing the property that Taq DNA polymerase does not have a 3'-5' proofreading function, controlling the frequency of random mutation under the conditions of high magnesium ion concentration (8mmol/L) and different concentrations of dNTP (wherein, the concentrations of dATP and dGTP are 1.5 mmol/L; the concentrations of dTTP and dCTP are 3.0mmol/L), introducing the random mutation into a target gene, and constructing a mutation library; template concentration A260 was 1000ng/mL, enzyme concentration was 5U/. mu.L, and primer concentration was 100. mu.M.

Error-prone PCR reaction (50. mu.l): 10 XPCR reaction buffer 5. mu.l, dNTP (2.5mM) 5. mu.l, MgCl₂(2.5mM) 5. mu.l, 1. mu.l of forward primer (HisG-F, SEQ ID NO:9), 1. mu.l of reverse primer (HisG-R, SEQ ID NO:10), 0.1. mu.l of DNA template (hisG/pUC57), 0.5. mu.l of Taq DNA polymerase, ddH₂O 32.4μl。

PCR procedure: pre-denaturation at 96 ℃ for 4 min; denaturation at 94 deg.C for 1min, annealing at 56 deg.C for 1min, extension at 75 deg.C for 2min, and 45 cycles; finally, extending for 15min at 75 ℃, and recovering a PCR product (the size of the product is 0.9kb) by adopting a glue recovery method; mu.l of the product was checked by electrophoresis on a 1% agarose gel and stored at-20 ℃ until use.

3. Construction of Gene mutant library of ATP phosphoribosyltransferase

The PCR product is digested by restriction enzymes Nco I and Hind III, then is subjected to ligation reaction with pTrc99A plasmid digested by Nco I and Hind III, and then the ligation product mixture is used for transforming Escherichia coli Top10, so that a large number of cloned transformants are obtained, and a transformed thallus mutation library is constructed.

4. Screening high-enzyme-activity mutant and preparation of recombinant strain thereof

About 5000 mutant clones were randomly picked from the transformed cell mutant pool, and inoculated into 5mL of LB medium containing 50. mu.g/mL of penicillin (Amp) using wild-type ZAS-004 as a control, cultured at 37 ℃ and 150rpm for 18 hours, and centrifuged at 10000rpm and 5mm to collect cells. After the supernatant is discarded, the supernatant is re-suspended in 1ml of PBS (pH value 7.5, 10mmol/L) solution at 4 ℃, 300V voltage is selected under the ice bath condition, ultrasonic crushing is carried out for 10min at 3s interval and 6s interval, the supernatant is taken as enzyme crude extract after centrifugation, and enzyme activity determination is carried out.

Detection of ATP phosphoribosyltransferase Activity: the decrease in the substrate 5-phosphoribosyl-alpha-pyrophosphate (PRPP) was used as a measurement marker. Definition of enzyme activity unit: the reduction in enzyme amount, defined as one enzyme activity unit (IU), under the enzymatic reaction conditions, corresponds to 1. mu. mol PRPP per minute. The specific operation is as follows: 5ml of reaction system is used as an enzyme activity determination system, which contains 500mmol/L PRPP, 5mmol/L glucose, 100mmol/L Tris-HCl (pH8.0) and 100. mu.l of crude enzyme solution. The enzyme activation reaction is carried out in a water bath at 37 ℃, the temperature is kept for 4h, and then the enzymatic hydrolysate is stopped at 70 ℃ for 10 min. Centrifuging at 3000rpm for 10min, and collecting supernatant. PRPP content was determined by HPLC.

The results show that: the enzyme activity of the highest mutant strain is 77.3IU/ml, and the enzyme activity of the contrast strain is 15.4 IU/ml. And (3) modifying HisG by error-prone PCR to obtain a mutant strain with the enzyme activity improved by about 5 times. Selecting mutant strain with highest enzyme activity, extracting plasmid and sequencing. The results show that: the gene sequence of the ATP phosphoribosyltransferase mutant is shown as SEQ ID NO. 11, and the corresponding amino acid sequence is shown as SEQ ID NO. 12. Compared with the sequence of the wild ATP phosphoribosyltransferase gene, 5 base point mutations occur: 396G/A, 591G/A,695A/T,754A/T, 822A/G; missense mutation at amino acid 3, wherein the mutation points are respectively as follows: H232L (histidine at position 232 changed to leucine), T252S (threonine at position 252 changed to serine), E271G (glutamic acid at position 271 changed to glycine). This mutant gene was designated as hisGM.

The hisGM/pTrc99A plasmid was prepared in the same manner as described above. Then, the hisGM/pTrc99A plasmid was transformed into the strain ZAS-003 to obtain a recombinant strain designated: ZAS-005(ZAS-001, hisGM/pTrc99, Δ purR).

Example 3

This example describes recombinant shake flask fermentation experiments, product assays, and histidine yields

1. Preparation of fermentation broth

Preparation of 5 XM 9 culture medium: about 800ml of double distilled water (ddH)₂O) to 64g of Na was added₂HPO₄·7H2O、15g KH₂PO₄、2.5g NaCl、5.0g NH₄Cl, dissolved and then water was added to 1000 ml. Sterilizing at 121 deg.C for 30 min. Respectively preparing 1M MgSO₄、1M CaCl₂20% glucose, and sterilized separately. Then, M9 culture medium was prepared as shown in Table 1, and 1000 Xtrace element solution was prepared as shown in Table 2.

TABLE 1M 9 culture solution composition

Composition (I)	Dosage (ml/L)
		5×M9	200
1M MgSO4	2
		1M CaCl2	0.1
20% glucose	20
		1000 x trace element solution	1
ddH2O	To 1000
		pH	6.9

TABLE 2.1000 Xtrace elements solution Components

Composition (I)	Dosage (g/L)
		CoCl2·6H2O	0.01
CuSO4·5H2O	0.01
		MnSO4·H2O	0.033
Fe SO4·7H2O	0.50
		ZnSO4·7H2O	0.38
H3BO3	0.01
		NaMoO4·2H2O	0.01
pH	3

2. Recombinant bacteria ZAS-005 and control ZAS-004, ZAS-003 and ZAS-001 shake flask fermentation test

Recombinant strains ZAS-005 freshly cultured on LB plate medium and monoclonal strains of control ZAS-004, ZAS-003 and parent strain ZAS-001 were inoculated into 3ml LB liquid medium tubes (13X 150mm) and cultured at 30 ℃ and 225rpm for about 8 hours. LB liquid Medium composition: 5g/l yeast powder, 10g/l peptone and 10g/l NaCl. Then, the seed culture was inoculated at 3% concentration into a 250ml shake flask containing 50ml of fermentation broth (M9 broth). Starting OD₆₀₀The culture was carried out at about 0.5, 37 ℃ and 225rpm for a fermentation period of 72 hours. At 24 hours, 48 hours, the broth pH was adjusted to 7.0 with 10M NaOH. According to the condition of sugar consumption of the fermentation liquid, 65 percent of glucose liquid is added in portions to maintain the glucose concentration at 20 g/L. After the fermentation, 1ml of the fermentation broth was collected and centrifuged. Histidine content was determined by HPLC.

3. HPLC determination of histidine content

Taking a sample, diluting by 10 times, shaking uniformly, and filtering by a 0.22um membrane.

Mobile phase: acetonitrile: water (containing 0.5% sodium dihydrogen phosphate) ═ 10:90

And (3) chromatographic column: c18

Wavelength: 210nm

Flow rate: 1ml/min

Sample introduction amount: 20 μ l

The peak time: 5.2min

4. Influence of recombinant bacterium plasmid transformation on yield of histidine in shake flask fermentation

The shake flask fermentation yields are shown in Table 3. The results show that: the control strains ZAS-001 and ZAS-003 are not detected, the yield of the recombinant bacteria ZAS-004 is low, and the yield of the recombinant bacteria ZAS-005 is obviously improved.

TABLE 3 Shake flask fermentation yield of pTrc-HisGM gene cassette integrated recombinant bacteria

Bacterial strain	Histidine yield (g/L)
		ZAS-005	1.9±0.3
ZAS-004	0.2±0.1
		ZAS-003	0.0±0.0
ZAS-001	0.0±0.0

The above results show that: overexpression of ATP phosphoribosyltransferase increases L-histidine production following deletion of the endogenous purine repressor; screening of mutants by error-prone PCR techniques also greatly increased the L-histidine yield, probably due to the increased enzyme activity resulting from the removal of the negative feedback restriction of L-histidine by the obtained enzyme mutants.

Example 4

This example describes the workup procedure for separation and purification of histidine.

Recombinant engineered strain ZAS-005 as production strain at 12M³The fermentation tank is used for fermentation according to a conventional process. After fermentation is finished, the product separation and purification process specifically comprises the following steps:

filtering the fermentation liquor by using a ceramic membrane, and carrying out solid-liquid separation. Heating the mother liquor to 70 ℃, adjusting the pH value to 7.5 by using 6mol/L HCl, adding activated carbon for decolorization, and stirring for 1h under heat preservation. The light transmittance after decolorization is more than 99%. Filtering with plate frame, ultrafiltering the filtrate, vacuum concentrating at 85 deg.C until a large amount of crystals appear, cold separating, centrifuging, collecting histidine to obtain refined histidine, and recovering mother liquor for reuse.

And (4) placing the refined histidine product in a double-cone rotary evaporation dryer for drying until the moisture reaches the standard. The product is as follows: and (3) obtaining the finished product of L-histidine hydrochloride.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> Nanjing Shobai Biotechnology Ltd

<120> method for producing histidine by microbial fermentation and application thereof

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 70

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gtaaaacgac ggccagtgga agttcctata ctttctagag aataggaact tcctcgtgaa 60

gaaggtgttg 70

<210> 2

<211> 80

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ggaaacagct atgaccatgc ctattccgaa gttcctattc tctagaaagt ataggaactt 60

ctgttacatt gcacaagata 80

<210> 3

<211> 1283

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gtaaaacgac ggccagtgga agttcctata ctttctagag aataggaact tcctcgtgaa 60

gaaggtgttg ctgactcata ccaggcctga atcgccccat catccagcca gaaagtgagg 120

gagccacggt tgatgagagc tttgttgtag gtggaccagt tggtgatttt gaacttttgc 180

tttgccacgg aacggtctgc gttgtcggga agatgcgtga tctgatcctt caactcagca 240

aaagttcgat ttattcaaca aagccgccgt cccgtcaagt cagcgtaatg ctctgccagt 300

gttacaacca attaaccaat tctgattaga aaaactcatc gagcatcaaa tgaaactgca 360

atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag 420

gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc 480

cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa 540

gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagc ttatgcattt 600

ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa 660

ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa 720

aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa 780

caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga 840

tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa 900

gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa 960

cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat 1020

agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag 1080

catccatgtt ggaatttaat cgcggcctcg agcaagacgt ttcccgttga atatggctca 1140

taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat gatgatatat 1200

ttttatcttg tgcaatgtaa cagaagttcc tatactttct agagaatagg aacttcggaa 1260

taggcatggt catagctgtt tcc 1283

<210> 4

<211> 1026

<212> DNA

<213> Escherichia coli (Escherichia coli)

<400> 4

atggcaacaa taaaagatgt agcgaaacga gcaaacgttt ccactacaac tgtgtcacac 60

gtgatcaaca aaacacgttt cgtcgctgaa gaaacgcgca acgccgtgtg ggcagcgatt 120

aaagaattac actactcccc tagcgcggtg gcgcgtagcc tgaaggttaa ccacaccaag 180

tctatcggtt tgctggcgac cagcagcgaa gcggcctatt ttgccgagat cattgaagca 240

gttgaaaaaa attgcttcca gaaaggttac accctgattc tgggcaatgc gtggaacaat 300

cttgagaaac agcgggctta tctgtcgatg atggcgcaaa aacgcgtcga tggtctgctg 360

gtgatgtgtt ctgagtaccc agagccgttg ctggcgatgc tggaagagta tcgccatatc 420

ccaatggtgg tcatggactg gggtgaagca aaagctgact tcaccgatgc ggtcattgat 480

aacgcgttcg aaggcggcta catggccggg cgttatctga ttgaacgcgg tcaccgcgaa 540

atcggcgtca tccccggccc gctggaacgt aacaccggcg caggccgcct tgccggtttt 600

atgaaggcga tggaagaagc gatgatcaag gtgccggaaa gctggattgt gcagggtgac 660

tttgaacctg aatccggtta tcgcgccatg cagcaaatcc tgtcgcagcc gcatcgccct 720

actgccgtct tctgtggtgg cgatatcatg gcaatgggcg cactttgtgc tgctgatgaa 780

atgggcctgc gcgtcccgca ggatgtttcg ctgatcggtt atgataacgt gcgcaacgcg 840

cgctatttta cgccggcgct gaccacgatc catcagccaa aagattcgct gggtgaaaca 900

gcgttcaaca tgctgttgga tcgtatcgtc aacaaacgtg aagaaccgca gtctattgaa 960

gtgcatccgc gcttgattga acgccgctcc gtggctgacg gcccgttccg cgactatcgt 1020

cgttaa 1026

<210> 5

<211> 68

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

taaaagatgt agcgaaacga gcaaacgttt ccactacaac tgtgtcacac gtaaaacgac 60

ggccagtg 68

<210> 6

<211> 69

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ttcaatagac tgcggttctt cacgtttgtt gacgatacga tccaacagca ggaaacagct 60

atgaccatg 69

<210> 7

<211> 299

<212> PRT

<213> Escherichia coli (Escherichia coli)

<400> 7

Met Thr Asp Asn Thr Arg Leu Arg Ile Ala Met Gln Lys Ser Gly Arg

1 5 10 15

Leu Ser Asp Asp Ser Arg Glu Leu Leu Ala Arg Cys Gly Ile Lys Ile

20 25 30

Asn Leu His Thr Gln Arg Leu Ile Ala Met Ala Glu Asn Met Pro Ile

35 40 45

Asp Ile Leu Arg Val Arg Asp Asp Asp Ile Pro Gly Leu Val Met Asp

50 55 60

Gly Val Val Asp Leu Gly Ile Ile Gly Glu Asn Val Leu Glu Glu Glu

65 70 75 80

Leu Leu Asn Arg Arg Ala Gln Gly Glu Asp Pro Arg Tyr Phe Thr Leu

85 90 95

Arg Arg Leu Asp Phe Gly Gly Cys Arg Leu Ser Leu Ala Thr Pro Val

100 105 110

Asp Glu Ala Trp Asp Gly Pro Leu Ser Leu Asn Gly Lys Arg Ile Ala

115 120 125

Thr Ser Tyr Pro His Leu Leu Lys Arg Tyr Leu Asp Gln Lys Gly Ile

130 135 140

Ser Phe Lys Ser Cys Leu Leu Asn Gly Ser Val Glu Val Ala Pro Arg

145 150 155 160

Ala Gly Leu Ala Asp Ala Ile Cys Asp Leu Val Ser Thr Gly Ala Thr

165 170 175

Leu Glu Ala Asn Gly Leu Arg Glu Val Glu Val Ile Tyr Arg Ser Lys

180 185 190

Ala Cys Leu Ile Gln Arg Asp Gly Glu Met Glu Glu Ser Lys Gln Gln

195 200 205

Leu Ile Asp Lys Leu Leu Thr Arg Ile Gln Gly Val Ile Gln Ala Arg

210 215 220

Glu Ser Lys Tyr Ile Met Met His Ala Pro Thr Glu Arg Leu Asp Glu

225 230 235 240

Val Ile Ala Leu Leu Pro Gly Ala Glu Arg Pro Thr Ile Leu Pro Leu

245 250 255

Ala Gly Asp Gln Gln Arg Val Ala Met His Met Val Ser Ser Glu Thr

260 265 270

Leu Phe Trp Glu Thr Met Glu Lys Leu Lys Ala Leu Gly Ala Ser Ser

275 280 285

Ile Leu Val Leu Pro Ile Glu Lys Met Met Glu

290 295

<210> 8

<211> 897

<212> DNA

<213> Escherichia coli (Escherichia coli)

<400> 8

atgacggata atacgcgtct gcgcatcgct atgcagaaat ctggccgtct gagcgacgat 60

tcccgtgaac tgctggctcg ctgtggtatc aaaattaacc tgcacaccca gcgtctgatc 120

gcgatggctg aaaacatgcc gattgacatc ctgcgtgtgc gcgacgatga cattccgggt 180

ctggttatgg atggcgtagt cgatctgggc atcattggtg aaaacgtcct ggaggaagaa 240

ctgctgaacc gtcgtgcgca gggtgaagat ccgcgttatt tcaccctgcg tcgtctggac 300

ttcggtggct gtcgtctgtc tctggcgact ccggttgatg aggcatggga tggtccgctg 360

agcctgaacg gcaaacgtat cgcgacctcc tatccgcatc tgctgaaacg ttatctggat 420

caaaaaggca tttctttcaa gagctgcctg ctgaatggtt ccgttgaagt agcgccgcgt 480

gctggtctgg cagatgctat ctgcgatctg gtaagcaccg gtgcaaccct ggaagcaaac 540

ggtctgcgtg aggtcgaagt gatctatcgt agcaaagcat gtctgatcca gcgtgatggt 600

gaaatggaag aaagcaaaca gcagctgatc gacaagctgc tgacccgtat ccagggtgtt 660

atccaggccc gtgaatccaa atatattatg atgcacgcac cgactgagcg tctggatgaa 720

gttatcgctc tgctgccagg tgcggaacgt ccgactattc tgccgctggc aggcgatcaa 780

cagcgtgttg cgatgcacat ggtctctagc gaaacgctgt tttgggaaac tatggaaaaa 840

ctgaaagcgc tgggcgcgtc ctctatcctg gtactgccta tcgaaaaaat gatggag 897

<210> 9

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

catgccatgg atgacggata atacgcgtct 30

<210> 10

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

cccaagctta ttactccatc attttttcga taggc 35

<210> 11

<211> 897

<212> DNA

<213> Escherichia coli (Escherichia coli)

<400> 11

atgacggata atacgcgtct gcgcatcgct atgcagaaat ctggccgtct gagcgacgat 60

tcccgtgaac tgctggctcg ctgtggtatc aaaattaacc tgcacaccca gcgtctgatc 120

gcgatggctg aaaacatgcc gattgacatc ctgcgtgtgc gcgacgatga cattccgggt 180

ctggttatgg atggcgtagt cgatctgggc atcattggtg aaaacgtcct ggaggaagaa 240

ctgctgaacc gtcgtgcgca gggtgaagat ccgcgttatt tcaccctgcg tcgtctggac 300

ttcggtggct gtcgtctgtc tctggcgact ccggttgatg aggcatggga tggtccgctg 360

agcctgaacg gcaaacgtat cgcgacctcc tatccacatc tgctgaaacg ttatctggat 420

caaaaaggca tttctttcaa gagctgcctg ctgaatggtt ccgttgaagt agcgccgcgt 480

gctggtctgg cagatgctat ctgcgatctg gtaagcaccg gtgcaaccct ggaagcaaac 540

ggtctgcgtg aggtcgaagt gatctatcgt agcaaagcat gtctgatcca acgtgatggt 600

gaaatggaag aaagcaaaca gcagctgatc gacaagctgc tgacccgtat ccagggtgtt 660

atccaggccc gtgaatccaa atatattatg atgctcgcac cgactgagcg tctggatgaa 720

gttatcgctc tgctgccagg tgcggaacgt ccgtctattc tgccgctggc aggcgatcaa 780

cagcgtgttg cgatgcacat ggtctctagc ggaacgctgt tttgggaaac tatggaaaaa 840

ctgaaagcgc tgggcgcgtc ctctatcctg gtactgccta tcgaaaaaat gatggag 897

<210> 12

<211> 299

<212> PRT

<213> Escherichia coli (Escherichia coli)

<400> 12

Met Thr Asp Asn Thr Arg Leu Arg Ile Ala Met Gln Lys Ser Gly Arg

1 5 10 15

Leu Ser Asp Asp Ser Arg Glu Leu Leu Ala Arg Cys Gly Ile Lys Ile

20 25 30

Asn Leu His Thr Gln Arg Leu Ile Ala Met Ala Glu Asn Met Pro Ile

35 40 45

Asp Ile Leu Arg Val Arg Asp Asp Asp Ile Pro Gly Leu Val Met Asp

50 55 60

Gly Val Val Asp Leu Gly Ile Ile Gly Glu Asn Val Leu Glu Glu Glu

65 70 75 80

Leu Leu Asn Arg Arg Ala Gln Gly Glu Asp Pro Arg Tyr Phe Thr Leu

85 90 95

Arg Arg Leu Asp Phe Gly Gly Cys Arg Leu Ser Leu Ala Thr Pro Val

100 105 110

Asp Glu Ala Trp Asp Gly Pro Leu Ser Leu Asn Gly Lys Arg Ile Ala

115 120 125

Thr Ser Tyr Pro His Leu Leu Lys Arg Tyr Leu Asp Gln Lys Gly Ile

130 135 140

Ser Phe Lys Ser Cys Leu Leu Asn Gly Ser Val Glu Val Ala Pro Arg

145 150 155 160

Ala Gly Leu Ala Asp Ala Ile Cys Asp Leu Val Ser Thr Gly Ala Thr

165 170 175

Leu Glu Ala Asn Gly Leu Arg Glu Val Glu Val Ile Tyr Arg Ser Lys

180 185 190

Ala Cys Leu Ile Gln Arg Asp Gly Glu Met Glu Glu Ser Lys Gln Gln

195 200 205

Leu Ile Asp Lys Leu Leu Thr Arg Ile Gln Gly Val Ile Gln Ala Arg

210 215 220

Glu Ser Lys Tyr Ile Met Met Leu Ala Pro Thr Glu Arg Leu Asp Glu

225 230 235 240

Val Ile Ala Leu Leu Pro Gly Ala Glu Arg Pro Ser Ile Leu Pro Leu

245 250 255

Ala Gly Asp Gln Gln Arg Val Ala Met His Met Val Ser Ser Gly Thr

260 265 270

Leu Phe Trp Glu Thr Met Glu Lys Leu Lys Ala Leu Gly Ala Ser Ser

275 280 285

Ile Leu Val Leu Pro Ile Glu Lys Met Met Glu

290 295

<210> 13

<211> 166

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

ttcgtgtcgc tcaaggcgca ctcccgttct ggataatgtt ttttgcgccg acatcataac 60

ggttctggca aatattctga aatgagctgt tgacaattaa tcatccggct cgtataatgt 120

gtggaattgt gagcggataa caatttcaca caggaaacag accatg 166

Claims (16)

1. A method for producing histidine by microbial fermentation, the method comprising:

A) culturing a microorganism in a fermentation medium, said microorganism comprising at least one genetic modification that increases the action of ATP Phosphoribosyl Transferase (hisG) in the microorganism; and

B) collecting the histidine produced from the culturing step A).

2. The method of claim 1, wherein the genetic modification to increase the action of ATP phosphoribosyltransferase in a microorganism is selected from the group consisting of a) an increase in the enzymatic activity of ATP phosphoribosyltransferase in a microorganism; and/or b) ATP phosphoribosyltransferase is overexpressed in the microorganism;

preferably, the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification that increases the action of ATP phosphoribosyltransferase in the microorganism.

3. The method of claim 2, wherein the microorganism is transformed with at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding an ATP phosphoribosyltransferase;

preferably, the nucleic acid sequence encoding ATP phosphoribosyltransferase contains at least one genetic modification that increases the enzymatic activity of ATP phosphoribosyltransferase; further preferably, the genetic modification comprises a modification in a nucleotide sequence corresponding to the amino acid sequence SEQ ID NO:12 at one or more of the following positions: histidine 232 substituted with leucine, threonine 252 substituted with serine, and glutamic acid 271 substituted with glycine; more preferably, the nucleic acid sequence encoding ATP phosphoribosyltransferase is SEQ ID NO 11;

preferably, the ATP phosphoribosyltransferase has an amino acid sequence that is at least about 30% identical, preferably at least about 50% identical, further preferably at least about 70% identical, further preferably at least about 80% identical, even further preferably at least about 90% identical, and most preferably at least about 95% identical to the amino acid sequence of SEQ ID NO. 12, wherein the ATP phosphoribosyltransferase has enzymatic activity; further preferably, the ATP phosphoribosyltransferase has the amino acid sequence of SEQ ID NO 12;

further preferably, the recombinant nucleic acid molecule has an increased copy number of a gene encoding an ATP phosphoribosyltransferase;

further preferably, the recombinant nucleic acid molecule comprises an endogenous native promoter or a promoter with a higher expression level than the endogenous native promoter; preferably, the promoter having a higher expression level than the endogenous native promoter is selected from the group consisting of HCE promoter, gap promoter, trc promoter, T7 promoter; further preferably, the promoter having a higher expression level than the endogenous native promoter is a trc promoter.

4. The method of claim 2, wherein the microorganism comprises at least one genetic modification to an endogenous native promoter of a gene encoding ATP phosphoribosyltransferase; preferably, the endogenous native promoter of the gene encoding ATP phosphoribosyltransferase is replaced by a promoter with a higher expression level; further preferably, the promoter having a higher expression level is selected from the group consisting of HCE promoter, gap promoter, trc promoter, T7 promoter; most preferably, the promoter with the higher expression level is the trc promoter.

5. The method of any one of claims 1 to 4, wherein the microorganism further comprises at least one genetic modification capable of reducing the action of the purine repressor PurR in the microorganism.

6. The method according to claim 5, wherein the microorganism is transformed with at least one recombinant nucleic acid molecule comprising at least one genetic modification capable of reducing the action of the purine repressor PurR in the microorganism;

preferably, the genetic modification which reduces the action of the purine repressor PurR in the microorganism is selected from the group consisting of a partial or complete deletion, or a partial or complete inactivation, of an endogenous gene encoding the purine repressor PurR in the microorganism and/or a partial or complete deletion, or a partial or complete inactivation, of an endogenous native promoter encoding the purine repressor PurR gene in the microorganism; more preferably, the genetic modification which reduces the action of the purine repressor protein PurR in the microorganism is a complete deletion, i.e.a deletion, of the endogenous gene coding for the purine repressor protein PurR in the microorganism.

7. The method of any one of claims 1-6, wherein the recombinant nucleic acid molecule is incorporated into a plasmid or the recombinant nucleic acid molecule is integrated into the genome of the microorganism.

8. The method of any one of claims 1-7, wherein expression of the recombinant nucleic acid molecule is inducible; preferably, the expression of the recombinant nucleic acid molecule is inducible by lactose.

9. The method of any one of claims 1-8, wherein the culturing step a) is performed at about 20 ℃ to about 45 ℃; preferably, from about 33 ℃ to about 37 ℃;

preferably, said culturing step A) is carried out at about pH4.5 to about pH 8.5; preferably at about pH6.7 to about pH 7.2;

preferably, the culturing step A) adopts a conventional fermentation medium;

further preferably, the fermentation medium comprises a carbon source; further preferably, the fermentation medium comprises a nitrogen source; further preferably, the fermentation medium comprises a carbon source and a nitrogen source; further preferably, the fermentation medium comprises a carbon source, a nitrogen source and inorganic salts;

still more preferably, the various carbon sources include organic carbon sources and/or inorganic carbon sources; preferably, the carbon source is selected from one or more of glucose, fructose, sucrose, galactose, dextrin, glycerol, starch, syrup and molasses; preferably, the concentration of the carbon source is maintained at about 0.1% to about 5%; still more preferably, the various nitrogen sources include organic nitrogen sources and/or inorganic nitrogen sources; preferably, the nitrogen source is selected from one or more of ammonia water, ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium acetate, sodium nitrate, urea, yeast extract, meat extract, peptone, fish meal, bean meal, malt, corn steep liquor and cotton seed meal;

preferably, the culturing step A) adopts a fed-batch fermentation method;

further preferably, the sugar-supplemented liquid contains glucose, preferably, the glucose concentration is 10% to 85% (w/v), and further preferably, the glucose concentration is 55% to 75% (w/v).

10. The method of any one of claims 1-7, wherein the collecting step B) comprises (a) precipitating histidine from the microorganism-removed fermentation broth; and/or (b) crystallizing histidine from the microorganism-depleted fermentation broth;

preferably, the collecting step B) further comprises a step of decolorizing the fermentation broth; further preferably, the decolorization step is performed before the fermentation broth is subjected to precipitation or crystallization, after the fermentation broth is subjected to one or more precipitation or crystallization resolubilizations; more preferably, the decolorizing step comprises activated carbon treatment and/or chromatographic decolorization.

11. The method of any one of claims 1-8 or any microorganism, wherein the microorganism is a bacterium, yeast, or fungus;

preferably, the microorganism is selected from bacteria or yeast;

further preferably, the bacteria are selected from bacteria of the genera Escherichia (Escherichia), Bacillus (Bacillus), Lactobacillus (Lactobacillus), Pseudomonas (Pseudomonas) or Streptomyces (Streptomyces); further preferably, the bacteria are selected from bacteria of the species Escherichia coli (Escherichia coli), Bacillus subtilis (Bacillus subtilis), Bacillus licheniformis (Bacillus licheniformis), Lactobacillus brevis (Lactobacillus brevis), Pseudomonas aeruginosa (Pseudomonas aeruginosa) or Streptomyces lividans (Streptomyces lividans); more preferably, the bacterium is escherichia coli; even more preferably, E.coli is selected from the group consisting of K-12, B and W strains; most preferably, Escherichia coli is K-12 strain;

further preferably, the yeast is selected from the group consisting of Saccharomyces (Saccharomyces), Schizosaccharomyces (Schizosaccharomyces), Candida (Candida), Hansenula (Hansenula), Pichia (Pichia), Kluyveromyces (Kluyveromyces), and Rhodofavus (Phaffia); more preferably, the yeast includes, but is not limited to, a yeast selected from the group consisting of Saccharomyces cerevisiae (Saccharomyces cerevisiae), Schizosaccharomyces pombe (Schizosaccharomyces pombe), Candida albicans (Candida albicans), Hansenula polymorpha (Hansenula polymorpha), Pichia pastoris (Pichia pastoris), Pichia canadensis (Pichia canadensis), Kluyveromyces marxianus (Kluyveromyces marxianus), or Phaffia rhodozyma (Phaffia rhodozyma);

preferably, the microorganism is a fungus; further preferably, the fungus is selected from the group consisting of fungi of the genera Aspergillus (Aspergillus), Absidia (Absidia), Rhizopus (Rhizopus), Chrysosporium (Chrysosporium), Neurospora (Neurospora) or Trichoderma (Trichoderma); more preferably, the fungus is selected from Aspergillus niger (Aspergillus niger), Aspergillus nidulans (Aspergillus nidulans), Absidia coerulea (Absidia coerulea), Rhizopus oryzae (Rhizopus oryzae), Rakennocardia serrulata (Chrysosporium lucknowense), Neurospora crassa (Neurospora crassa), Neurospora intermedius (Neurospora intermedia) or Trichoderma reesei (Trichoderma reesei).

12. An ATP phosphoribosyltransferase with higher enzymatic activity, said enzyme having the amino acid sequence of SEQ ID NO: 4.

13. A nucleic acid molecule encoding the ATP phosphoribosyltransferase of claim 12, having the sequence of SEQ ID NO: 3.

14. A vector comprising the nucleic acid molecule of claim 13.

15. A microorganism comprising the vector of claim 14.

16. A microorganism comprising in its genome the nucleic acid molecule of claim 13.

Images