CN117844706A

CN117844706A - Ergothioneine producing strain fermentation medium

Info

Publication number: CN117844706A
Application number: CN202410079306.4A
Authority: CN
Inventors: 王金刚; 任亮; 梁岩; 步绪亮; 韦炎龙
Original assignee: Shanghai Banglin Biotechnology Co ltd
Current assignee: Shanghai Banglin Biotechnology Co ltd
Priority date: 2024-01-19
Filing date: 2024-01-19
Publication date: 2024-04-09

Abstract

The invention discloses a fermentation medium of ergothioneine production bacteria, which comprises 0.5-10g/L methyl histidine. Experiments show that the addition of methyl histidine in the culture medium can obviously improve the yield of ergothioneine produced by a fermentation method.

Description

Ergothioneine producing strain fermentation medium

Technical Field

The invention belongs to the field of fermentation engineering, and relates to a fermentation medium of ergothioneine production bacteria and a method for producing ergothioneine by a fermentation method.

Background

Ergothioneine (EGT), known as 2-mercaptohistidine trimethylinner salt, is a rare amino acid found in nature and was isolated from ergot in 1909 and is therefore known. Of the general antioxidants, ergothioneine is particularly unique in that it can sequester heavy metal ions and protect erythrocytes in the body from free radical damage. The ergothioneine has the property of coenzyme, participates in various physiological and biochemical activities of human body, has very strong oxidation resistance, can obviously improve the activity of epidermal cells, has an anti-aging effect, can reduce the generation of melatonin, prevent skin surface aging, can inhibit saccharification reaction of skin protein, has obvious effect of improving skin, and has the effect of promoting hair growth.

Ergothioneine is currently prepared mainly by chemical synthesis methods and biological synthesis methods, wherein the chemical synthesis methods have the advantages of complex process, low product yield, high synthesis cost and difficult industrialization. The biosynthesis method is divided into two types, one is a fungus hypha fermentation method and the other is a genetic engineering fungus catalytic synthesis method. In the method for preparing the L-ergothioneine by fermenting mushroom mycelia, the yield of mycelia is low, byproducts in fermentation liquor are more, the later extraction is affected, and the high-purity ergothioneine cannot be prepared on a large scale.

Compared with the extraction of ergothioneine from mycelium, the genetically engineered metabolic bacteria has higher yield, lower fermentation cost, fewer byproducts and lower extraction difficulty, and has industrialized prospect. For example, WO2017150304A1 discloses that Streptomyces lividans Streptomyces lividans is fermented for 7 days and that ergothioneine yield is 900mg/L; CN111534535A discloses that rhodotorula glutinis is used as a host to express exogenous genes, and the yield of ergothioneine reaches 1.5g/L.

The inventor also takes escherichia coli as a host, successfully constructs ergothioneine production bacteria, can convert and generate the ergothioneine through fermenting and catalyzing substrates L-histidine, L-methionine (L-methionine) and L-cysteine, and after 4 days of conversion, the yield of the ergothioneine reaches 5.1g/L, and is shown in patent document CN202310214002.X.

The synthesis of ergothioneine in microorganisms requires 2 key enzymes, namely, ergothioneine synthase 1 (NsEgt 1) and ergothioneine synthase 2 (NcEgt 2), and the direct fermentation method is to metabolize L-histidine, L-methionine and L-cysteine by microorganisms to produce ergothioneine; at present, the direct fermentation method of ergothioneine is mainly optimized aiming at strains and culture mediums, and systematic optimization investigation on fermentation conditions is rarely performed.

Disclosure of Invention

In order to improve the productivity of producing ergothioneine by a genetically engineered metabolic bacteria fermentation method, a great deal of research and exploration are also carried out on the composition of a culture medium. For example, we have studied compounds and proteins capable of promoting histidine metabolism, as well as increasing the activity of NsEgt1 and/or NcEgt2 enzymes, starting from metabolic pathways of histidine bioconversion, and found that some compounds of specific structure have the effect of promoting ergothioneine biosynthesis, with the methyl histidine promoting effect being particularly pronounced. Based on the discovery, the invention comprises the following technical scheme:

in a first aspect, the invention provides the use of methylhistidine in the preparation of a fermentation medium for ergothioneine production bacteria.

The ergothioneine-producing strain is a microorganism capable of catalyzing L-histidine to synthesize ergothioneine, particularly a microorganism capable of catalyzing histidine, methionine and cysteine to synthesize ergothioneine, preferably an engineering strain of Escherichia coli, more preferably an engineering strain of Escherichia coli reported in patent document CN202310214002.X.

The E.coli engineering bacteria reported in CN202310214002.X are engineering bacteria BL9-M1278 or pTrc99a-trc-NsEgt1-trc-NcEgt2/Ecoli BW 25113-DeltaYS constructed by taking E.coli BW25113 as chassis bacteria.

In one embodiment, the methylhistidine is used in an amount of about 0.5 to 10g/L, for example about 0.8 to 9g/L, preferably about 1.0 to 8g/L, more preferably about 1.2 to 7g/L, more preferably about 1.5 to 6g/L, more preferably about 1.8 to 5g/L, and more preferably about 2.0g/L, in the ergothioneine-producing strain fermentation medium.

In a second aspect, the invention provides a fermentation medium of ergothioneine, which is a medium formed by adding 0.5-10g/L methyl histidine to a common fermentation medium (common fermentation medium) of ergothioneine.

For example, the above-mentioned usual fermentation medium (ordinary fermentation medium) may contain the following components: 10g/L to 40g/L of glucose, 2g/L to 20g/L of peptone, 2g/L to 40g/L of yeast extract, 10g/L to 40g/L of corn steep liquor, 0.1 g/L to 1g/L of magnesium sulfate heptahydrate and 7g/L to 40g/L of buffer salt, wherein the buffer salt comprises the following components, preferably consists of the following components: 1-10g/L of betaine hydrochloride, 0.2-1g/L of monopotassium phosphate, 0.5-5g/L of dipotassium phosphate, 5-30g/L of ammonium sulfate, 0.2-1g/L of diammonium hydrogen citrate and 0.1-1.0g/L of ferric ammonium citrate.

Furthermore, the ergothioneine-producing strain fermentation medium can also comprise 1.5-10g/L of ergothioneine synthesis precursor amino acid, wherein the precursor amino acid comprises the following components, preferably consists of the following components: l-histidine 0.5-5g/L, L-methionine 0.5-5g/L and L-cysteine 0.5-5g/L.

The ergothioneine-producing strain fermentation medium can also comprise 0.003-0.03g/L of vitamins which comprise the following components, preferably consist of the following components: VB 1.001-0.01 g/L, VB, 0.001-0.01g/L and VB120.001-0.01g/L.

In one embodiment, the ergothioneine-producing strain fermentation medium described above may further comprise 30-200mg/L isopropyl-beta-D-thiogalactoside.

Preferably, the ergothioneine producing strain fermentation culture can also comprise 0.2-2ml/L of salt solution, wherein the salt solution comprises the following components: feSO ₄ ·7H ₂ O 10g/L，CuCl ₂ 0.2g/L，MnSO ₄ ·5H ₂ O 1g/L，CaCl ₂ ·2H ₂ O 0.2g/L，(NH ₄ ) ₆ Mo ₇ O ₂₄ 2g/L。

In a third aspect, the invention provides the use of the ergothioneine-producing strain fermentation medium described above in the fermentative production of ergothioneine.

In one embodiment, the fermentation further comprises a feed medium, wherein the feed medium comprises the following components: glucose about 700g/L, methyl histidine about 30g/L, betaine hydrochloride about 5g/L, potassium dihydrogen phosphate about 2g/L, ammonium sulfate about 10g/L, yeast extract about 5g/L, peptone about 5g/L, corn steep liquor about 20g/L, glycerol about 100g/L, VB1 about 0.01g/L, VB6 about 0.01g/L, VB12 about 0.01g/L, L-histidine about 10g/L, L-methionine about 30g/L, L-cysteine about 30g/L, and saline about 1ml/L, wherein the composition of the saline solution is as follows: feSO ₄ ·7H ₂ O about 10g/L, cuCl ₂ About 0.2g/L MnSO ₄ ·5H ₂ O about 1g/L, caCl ₂ ·2H ₂ O about 0.2g/L, (NH) ₄ ) ₆ Mo ₇ O ₂₄ About 2g/L.

The terms "about," "approximately," or "about," when used herein in reference to a numerical feature, mean that the number represented may have an error range or float range of + -10%, + -9%, + -8%, + -7%, + -6%, or + -5%. In addition, unless otherwise defined, the scope of the terms presented herein includes the terms and any number within the scope.

Further, the fermentation also comprises a strain activation culture medium and a seed culture, and for the escherichia coli engineering bacteria, the composition of the activation culture medium is as follows: 15g/L peptone, 5g/L yeast extract and 10g/L sodium chloride.

For E.coli engineering bacteria, for example, the seed medium composition is as follows: 24g/L peptone, 12g/L yeast extract, 8g/L glycerol, 16g/L dipotassium hydrogen phosphate and 6g/L potassium dihydrogen phosphate.

pH control in fermentation process: the pH is controlled to be 6.8-7.2 in the whole process until the fermentation is finished.

And (3) temperature control in the fermentation process: controlling the temperature of the fermentation at 37 ℃ for 0-12h, controlling the temperature of the fermentation at 30 ℃ for 12-24 h, controlling the temperature of the fermentation at 28 ℃ for 24-48 h, controlling the temperature of the fermentation at 25 ℃ for 48-72 h, and ending the fermentation.

According to the invention, by adding methyl histidine into the ergothioneine producing strain fermentation medium, not only is fermentation proliferation of the ergothioneine producing strain unaffected, but also biosynthesis capacity of producing strain for catalyzing histidine, methionine and cysteine to convert into ergothioneine can be remarkably improved. The yield of the product ergothioneine is improved from the initial 1.1g/L to 7.2g/L, and the yield is improved by 554%, thereby being beneficial to the realization of the industrial production of the catalytic synthesis of the ergothioneine by genetic engineering bacteria.

Drawings

FIG. 1 is a bar graph of the effect of single factor on the content of ergothioneine and the growth of the cells produced by the fermentation process for examining the effect of different nitrogen sources.

FIG. 2 is a bar graph showing the relationship between the amount of isopropyl- β -D-thiogalactoside added and the content of ergothioneine produced by fermentation and the growth of bacterial cells.

FIG. 3 is a bar graph showing the relationship between the amount of methylhistidine added and the ergothioneine content produced by fermentation and the growth of the cells.

FIG. 4 shows the course of the concentration and variation of the content of ergothioneine produced by fermentation.

Detailed Description

Optimization of the culture medium is an essential task in order to increase the production level of fermentation products such as ergothioneine. The culture medium mainly comprises a carbon source, a nitrogen source, inorganic salts and the like, so that the design and optimization of the culture medium are mainly centered on the carbon source, the nitrogen source, the inorganic salts and the like. For microbial fermentation for the production of specific generation of elyses, it is also necessary to study the effect of certain stimulus factors.

Common nitrogen sources can be divided into organic nitrogen sources and inorganic nitrogen sources. The organic nitrogen source is rich in nutrition, so that microorganisms often show the characteristic of vigorous growth and rapid growth of thallus concentration in a culture medium containing the organic nitrogen source, and the protein is taken as the nitrogen source to often promote enzyme production. Microorganisms typically take up inorganic nitrogen sources such as ammonium salts, nitrates, ammonia, etc. faster than organic nitrogen sources, but often cause large changes in pH. The proper use of these physiological acid-base substances has a positive effect on stabilizing and regulating the pH of the fermentation process, and therefore, in the production of many microbial enzymes, organic nitrogen sources and inorganic nitrogen sources are often used in combination.

Among the commonly used carbon sources, glucose is the most readily available sugar for microorganisms, often as a major component of the medium, but glucose repression effects occur at too high concentrations, and is therefore often used in combination with other carbon sources in the batch fermentation of microorganisms. Besides saccharides, alcohols such as glycerol can also be used as a carbon source and energy source for microorganisms.

Inorganic salts are also essential for the growth and metabolism of microorganisms, such as: phosphate is not only a phosphorus source, but also can regulate the osmotic pressure of cells, and can also play a role of a buffering agent for stabilizing the pH of fermentation liquor.

The inventor carries out single-factor tests on carbon sources, nitrogen sources, inorganic salts and certain excitation factors one by one, examines the influence of the single-factor tests on the L-histidine conversion of the production bacteria to produce ergothioneine, and optimizes the composition of the culture medium by applying common mathematical methods such as orthogonal design, uniform design and the like.

Based on the research of histidine bioconversion metabolic pathway, the invention screens out fermentation medium additives capable of improving histidine metabolic efficiency, effectively promotes ergothioneine biosynthesis, and methyl histidine is one of them.

Methyl histidine is a natural non-protein amino acid, known as 1-methyl-L-histidine, CAS number: 332-80-9, the structural formula is as follows:

to describe this bioconversion enhancing effect, "methylhistidine" may be referred to herein as "medium additive" or simply "additive".

Methyl histidine has a substrate-like effect, i.e., is similar to L-histidine, in addition to the effect of the stimulating factor, and its principle is to be studied further.

The methyl histidine can be applied to fermentation culture of escherichia coli engineering bacteria and synthesis of ergothioneine of other types of microorganisms. The other types of microorganisms comprise engineering bacteria with ergothioneine synthesis capability, such as mycobacterium, streptomyces, mould, yeast and the like.

Preferably, the microorganism is an E.coli engineering bacterium, for example, an E.coli engineering bacterium reported in patent document CN202310214002.X. One advantage of using E.coli as ergothioneine-producing bacteria to catalyze histidine, methionine and cysteine to synthesize ergothioneine is that most of ergothioneine produced by fermentation of the strain can be secreted into fermentation liquor, so that the ergothioneine as an extracellular product is very beneficial to extraction and purification of the product because wall breaking treatment of fermentation thalli is not needed, and complex post-treatment steps such as separation and extraction are omitted, thereby reducing the production cost.

According to the research, after methyl histidine is added into the escherichia coli engineering bacteria fermentation culture solution, the biosynthesis capacity of the strain for catalyzing histidine and/or converting histidine, methionine and cysteine into ergothioneine is obviously improved.

As used herein, the term "(ergothioneine synthesis ability) as used above is increased," "elevated," or "enhanced" means at least a 20% increase compared to a reference level, such as at least about 30%, at least about 50%, or at least about 80%, at least about 1-fold, at least about 2-fold, or at least about 3-fold, or at least about 4-fold increase compared to a reference level.

In a specific embodiment of the invention, the methyl histidine content in the fermentation medium is 0.5-10g/L, i.e. the lower limit of the methyl histidine content in the fermentation medium is 0.5g/L, preferably 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.21.3, 1.4 or 1.5g/L; the upper limit is 10g/L, preferably 9.5, 9.0, 8.5, 8.0, 7.8, 7.5, 7.2, 7.0, 6.8, 6.5, 6.2, 6.0, 5.8, 5.5, 5.2 or 5.0g/L. If the content of methyl histidine is less than 0.5g/L, the synthesis capacity of ergothioneine is not obviously improved, and the excitation factor is not played; on the other hand, if the content of methylhistidine is more than 10g/L, the production cost of ergothioneine is too high, and even the substrate concentration is excessive, so that the yield of ergothioneine is improved and the production cost cannot reach the expected balance.

isopropyl-beta-D-thiogalactoside also has a similar "stimulating factor" effect in the fermentation medium and can be used in an amount of 30-200mg/L, preferably 35-180mg/L, 40-150mg/L, 45-120mg/L, more preferably 48-96mg/L.

The addition of methyl histidine and/or isopropyl-beta-D-thiogalactoside as "stimulating factors" can effectively improve the biosynthesis capacity of the strain for catalyzing histidine and/or converting histidine, methionine and cysteine into ergothioneine. When the strain is E.coli, the composition as a preferred fermentation medium formulation is as follows: 10g/L to 40g/L of glucose, 1g/L to 10g/L of methyl histidine, 1g/L to 10g/L of betaine hydrochloride, 0.2g/L to 1g/L of monopotassium phosphate, 0.5g/L to 5g/L of dipotassium phosphate, 5g/L to 30g/L of ammonium sulfate, 0.2g/L to 1g/L of diammonium citrate, 0.1 g/L to 1.0g/L of ferric ammonium citrate, 2g/L to 20g/L of peptone, 2g/L to 40g/L of yeast extract, 10g/L to 40g/L of corn steep liquor, 0.1 g/L to 1g/L of magnesium sulfate heptahydrate, 0.5g/L to 5g/L of L-histidine, 0.5g/L to 5g/L of L-methionine, 0.001 g/L to 0.01g/L of VB, 0.001 g/L to 0.0.01g/L, VB 12.001-0.01 g/L, salt solution 0.2-2ml/L and isopropyl-beta-D-thiogalactoside 48-96mg/L. Wherein, the composition of the salt solution is as follows: feSO ₄ ·7H ₂ O10g/L，CuCl ₂ 0.2g/L，MnSO ₄ ·5H ₂ O 1g/L，CaCl ₂ ·2H ₂ O 0.2g/L，(NH ₄ ) ₆ Mo ₇ O ₂₄ 2g/L。

In the description of the technical solutions of the present invention, terms such as "a and/or B", "the term" and/or "used in" a and/or B "are intended to include both a and B; a or B; a (alone); and B (alone). Likewise, the term "and/or" as used in phrases such as "A, B and/or C" is intended to encompass each of the following embodiments: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; a (alone); b (alone); c (alone).

The fermentation medium of the present invention is preferably used for fermentation of an engineering bacterium of escherichia coli reported in patent document cn202310214002.X, which can be constructed by a method comprising the steps of:

A. taking escherichia coli BW25113, MG1655 or W3110 as chassis bacteria, knocking out or weakening an L-cysteine degradation pathway to obtain a strain A;

B. the strain A is used for over-expressing the Neurospora sp. The ergothioneine synthetase 1 or the mutant thereof from Neurospora sp. And the ergothioneine synthetase 2 or the NcEgt2 from Neurospora crassa Neurospora crassa, and positive clones are screened to obtain the engineering bacteria of the escherichia coli for producing the ergothioneine.

Alternatively, step A is the knockout of the ygeA gene (GI: 446771403) and the sseA gene (GI: 446030771); and/or

Step B is to transform the expression plasmid of the ergothioneine synthase 1, namely NsEgt1 or a mutant thereof and the expression plasmid of the ergothioneine synthase 2, namely NcEgt2 into cells of the strain A.

Wherein the nucleotide sequence of the encoding gene of the ergothioneine synthase 1, namely the NsEgt1, is SEQ ID NO 1 in the sequence table of the patent document; the nucleotide sequence of the coding gene of the ergothioneine synthetase 1 mutant is SEQ ID NO 5 in the sequence table of the patent document; the nucleotide sequence of the encoding gene of the ergothioneine synthase 2, namely NcEgt2, is SEQ ID NO 3 in the sequence table of the patent document.

Engineering bacteria BL9-M1278 or pTrc99 a-trc-NcEgt 1-trc-NcEgt2/Ecoli BW 25113-DeltaYS constructed with E.coli BW25113 as Chaetobacter are preferred.

Wherein the NsEgt1 amino acid sequence in the strain pTrc99a-trc-NsEgt1-trc-NcEgt2/Ecoli BW 25113-DeltaYS is SEQ ID NO. 2 in the sequence table of the patent document, and the NsEgt1 amino acid sequence in the strain BL9-M1278 is SEQ ID NO. 6 in the sequence table of the patent document.

As is well known to those skilled in the art, taking e.coli engineering bacteria as an example, the fermentation process of ergothioneine using the fermentation medium is approximately as follows:

transferring the strain from the glycerol tube to a shake flask to obtain seed liquid;

inoculating the cultured seed liquid to a fermentation tank, and initially stirring the fermentation tank at 400rpm; the culture temperature is 25-37 ℃; controlling the pH value to be 6.8-7.2 by ammonia water; controlling the dissolved oxygen by 20% -30% through stirring and ventilation; feeding the feed medium is started when the dissolved oxygen is higher than 50%, and the fermentation is finished.

According to the invention, by adding methyl histidine and/or isopropyl-beta-D-thiogalactoside, an organic nitrogen source is added, so that thalli grow rapidly, meanwhile, the culture time at 37 ℃ is prolonged, the growth rate of thalli is improved, dissolved oxygen is controlled to be 20% -30%, oxygen is rapidly supplemented for the growth of escherichia coli, the temperature is reduced in the fermentation process, the correct folding of ergothioneine synthase is facilitated, and the synthesis efficiency of ergothioneine is improved by adding methyl histidine.

The present invention will be described in further detail with reference to specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

The amounts, amounts and concentrations of various substances are referred to herein, wherein the percentages refer to percentages by mass unless otherwise specified.

Example 1

The experimental strain is the escherichia coli engineering bacterium BL9-M1278 reported in the patent document CN202310214002.X, and is constructed and stored by Shanghai Pont Biotechnology Co., ltd, and can be obtained by any unit and person for verifying the invention, but is not allowed to be used for other purposes by Shanghai Pont Biotechnology Co., ltd, including development and utilization, strain improvement, scientific research and teaching.

Activating the strain for 18h based on 37 DEG C220 rpm, obtaining an activated seed liquid, inoculating the activated seed liquid into a seed culture medium according to an inoculum size of 1v/v%, culturing for 10h at 37 DEG C220 rpm, and inoculating the activated seed liquid into a fermentation tank according to an inoculum size of 5 v/v%.

Fermenting in a fermentation tank: the inoculation amount is 5v/v%, the fermentation volume is 2L/5L, the initial rotating speed is 400rpm, the initial ventilation amount is 120L/h, the culture temperature is 30 ℃, the pH is controlled to 7.0, the dissolved oxygen is maintained to 20% -30%, the dissolved oxygen is higher than 50%, the feeding is started, and the feeding and the dissolved oxygen are controlled in a combined way.

And after the fermentation is finished for 72 hours, detecting the content of ergothioneine by high performance liquid chromatography.

Ergothioneine standard was purchased from the company alaa Ding Huaxue reagent.

Ergothioneine HPLC detection conditions: agilent high performance liquid chromatograph 1260infinity II,SB-AQ 250mm×4.6mm,5 μm; a detection wavelength of 258nm; mobile phase a: b (95:5); operating temperature 40 ℃; the flow rate is 0.5ml/min; mobile phase a:0.1% ammonium acetate; mobile phase B: acetonitrile.

Amino acid HPLC detection conditions: waters Symmetry (250 x 4.6mm,5 um) column, flow rate 1ml/min, wavelength: 338nm, column temperature: 35 ℃, 20ul of sample injection amount, mobile phase A: b (40:60)

Mobile phase a phase: 2.72g/L sodium acetate, 0.018% triethylamine, 0.3% tetrahydrofuran (pH 7.20).

Mobile phase B phase: 50% acetonitrile, 50% methanol.

Initial fermentation medium: glucose 20g/L, potassium dihydrogen phosphate 0.5g/L, dipotassium hydrogen phosphate 2g/L, ammonium sulfate 5g/L, diammonium hydrogen citrate 0.5g/L, ferric ammonium citrate 0.5g/L, yeast extract 2g/L, magnesium sulfate heptahydrate 0.5g/L, L-methionine 2g/L, L-cysteine 2g/L, VB 1.005 g/L, VB 6.005 g/L, VB 12.005 g/L, saline solution 1ml/L, isopropyl-beta-D-thiogalactoside 48mg/L, and water as solvent.

Initial feed medium: 700g/L of glucose, 2g/L of monopotassium phosphate, 5g/L of ammonium sulfate, 2g/L of yeast extract, 100g/L of glycerin, 0.01g/L of VB12, 30g/L of L-methionine, 30g/L of L-cysteine and 1ml/L of saline solution.

The composition of the salt solution is as follows: feSO ₄ ·7H ₂ O 10g/L，CuCl ₂ 0.2g/L，MnSO ₄ ·5H ₂ O 1g/L，CaCl ₂ ·2H ₂ O0.2g/L，(NH ₄ ) ₆ Mo ₇ O ₂₄ 2g/L (hereinafter salt solutions are all of this formula).

After 72 hours of fermentation, the OD600 is 52.1, and the content of ergothioneine is 1.1g/L.

Example 2

Nitrogen source single factor experiment, based on the initial fermentation medium without any nitrogen source in example 1, 1% of different nitrogen sources are added to obtain shake flask fermentation medium, and the influence of different nitrogen sources on thallus growth and ergothioneine yield is examined. The nitrogen source is selected from yeast powder, fish peptone, soybean peptone, tryptone, ammonium sulfate, ammonium chloride, and corn steep liquor.

BL9-M1278 glycerol tubes are inoculated into 50ml LB shake flasks according to 1v/v percent of inoculum size, 37 DEG C230 rpm, cultured for 12 hours to obtain seed liquid, and then inoculated into shake flask fermentation media according to 1v/v percent of inoculum size, 37 DEG C230 rpm, cultured for 72 hours, and the content of ergothioneine in fermentation OD600 and fermentation supernatant is measured.

The results of fermentation of different nitrogen sources are shown in figure 1, and the content of ergothioneine is as follows in sequence: fish peptone, ammonium sulfate, corn steep liquor, yeast extract, tryptone, soybean peptone, and ammonium chloride.

From the fermentation results, the organic nitrogen source is superior to the inorganic nitrogen source in growth, wherein the fish peptone is most beneficial to the synthesis of ergothioneine, and in order to ensure the bacterial amount and the yield, the influence of the fish peptone, ammonium sulfate and corn steep liquor on the fermentation is examined.

Example 3

Based on the initial fermentation medium in example 1, the effect of different addition amounts on the growth of thalli and the yield of ergothioneine was examined in the single factor experiment of isopropyl-beta-D-thiogalactoside (IPTG), and the addition amounts of isopropyl-beta-D-thiogalactoside were 0mg/L,24mg/L,48mg/L,96mg/L and 240mg/L, respectively.

The fermentation results of different isopropyl-beta-D-thiogalactoside addition amounts are shown in figure 2, the micro-expression can be realized without adding isopropyl-beta-D-thiogalactoside, the addition amount of 240mg/L seriously inhibits the growth of thalli, the OD600 is only 3.2, and the content of ergothioneine of the addition amount of 48mg/L is the highest and reaches 0.472g/L.

Example 4

Methyl histidine single factor experiment, based on the initial fermentation medium in example 1, examined the influence of different addition amounts on cell growth and ergothioneine yield, the methyl histidine addition amounts were 0g/L,1g/L,5g/L,10g/L, respectively.

BL9-M1278 glycerol tubes stored by the company are inoculated into 50ml LB shake flasks according to an inoculum size of 1%, the mixture is cultured for 12 hours at 37 DEG C230 rpm, seed liquid is obtained, then the seed liquid is cultured for 72 hours according to an inoculum size of 1% to shake flask fermentation medium at 37 DEG C230 rpm, and the methyl histidine addition amounts are 0g/L,1g/L,5g/L and 10g/L respectively, and the content of ergothioneine in the fermentation shake flasks OD600 and the content of ergothioneine in the fermentation supernatant are measured.

The results of the fermentation of methyl histidine at different concentrations are shown in FIG. 3, and 5g/L methyl histidine is added, with the highest content of ergothioneine being 0.604g/L.

Example 5

Activating strain BL9-M1278 with an activating culture medium for 18h to obtain an activating seed liquid, inoculating the activating seed liquid into the seed culture medium according to an inoculum size of 1v/v%, culturing at 37 DEG C220 rpm for 10h, and inoculating into a fermentation tank according to an inoculum size of 5 v/v%.

Fermenting in a fermentation tank: the inoculation amount is 5 percent, the fermentation volume is 2L/5L, the initial rotating speed is 400rpm, the initial ventilation amount is 120L/h, the culture temperature is 30 ℃, the pH is controlled to 7.0, the dissolved oxygen is maintained to be 20-30 percent, the dissolved oxygen is higher than 50 percent, the feeding is started, and the feeding and the dissolved oxygen are controlled in a combined way.

Fermentation medium: glucose 20g/L, betaine hydrochloride 5g/L, potassium dihydrogen phosphate 0.5g/L, dipotassium hydrogen phosphate 2g/L, ammonium sulfate 5g/L, diammonium hydrogen citrate 0.5g/L, ferric ammonium citrate 0.5g/L, yeast extract 5g/L, fish peptone 10g/L, corn steep liquor 20g/L, magnesium sulfate heptahydrate 0.5g/L, L-methionine 2g/L, L-cysteine 2g/L, VB 1.005 g/L, VB60.005g/L, VB 12.005 g/L, saline solution 1ml/L, isopropyl-beta-D-thiogalactoside 48mg/L;

feed medium: 700g/L of glucose, 5g/L of betaine hydrochloride, 2g/L of monopotassium phosphate, 5g/L of ammonium sulfate, 5g/L of yeast extract, 10g/L of fish peptone, 20g/L of corn steep liquor, 100g/L of glycerin, 0.01g/L of VB1, 0.01g/L of VB6, 0.01g/L of VB12, 30g/L of L-methionine, 30g/L of L-cysteine and 1ml/L of salt solution.

At the end of 72h fermentation, the OD600 was 100.5, and the ergothioneine content was increased by 18% compared to example 1.

Example 6

Activating BL9-M1278 strain with activating culture medium for 18 hr to obtain activating seed liquid, inoculating the activating seed liquid into the seed culture medium in 1% inoculation amount, culturing at 37 deg.c and 220rpm for 10 hr, and inoculating to fermentation tank in 5% inoculation amount.

Fermentation medium: glucose 20g/L, betaine hydrochloride 5g/L, monopotassium phosphate 0.5g/L, dipotassium phosphate 2g/L, ammonium sulfate 5g/L, diammonium hydrogen citrate 0.5g/L, ferric ammonium citrate 0.5g/L, yeast extract 5g/L, fish peptone 10g/L, corn steep liquor 20g/L, magnesium sulfate heptahydrate 0.5g/L, L-histidine 2g/L, L-methionine 2g/L, L-cysteine 2g/L, VB10.005g/L, VB 6.005 g/L, VB 12.005 g/L, saline solution 1ml/L, isopropyl-beta-D-thiogalactoside 48mg/L, and water as solvent;

feed medium: 700g/L of glucose, 5g/L of betaine hydrochloride, 2g/L of monopotassium phosphate, 5g/L of ammonium sulfate, 5g/L of yeast extract, 10g/L of fish peptone, 20g/L of corn steep liquor, 100g/L of glycerin, 0.01g/L of VB1, 0.01g/L of VB6, 0.01g/L of VB12, 30g/L of L-histidine, 30g/L of L-methionine, 30g/L of L-cysteine and 1ml/L of saline solution.

After 72 hours of fermentation, the OD600 is 110.6, and compared with example 1, the content of ergothioneine is improved by 242%.

Example 7

Fermenting in a fermentation tank: the inoculation amount is 5%, the fermentation volume is 2L/5L, the initial rotating speed is 400rpm, the initial ventilation amount is 120L/h, the culture temperature is 30 in 0-24 h, the culture temperature is 28 in 24-72 h, the pH is controlled at 7.0, the dissolved oxygen is maintained to be 20-30%, the dissolved oxygen is higher than 50%, the feeding is started, and the feeding and the dissolved oxygen combined control are performed.

Fermentation medium: glucose 20g/L, methylhistidine 2g/L, betaine hydrochloride 5g/L, monopotassium phosphate 0.5g/L, dipotassium phosphate 2g/L, ammonium sulfate 5g/L, diammonium citrate 0.5g/L, ferric ammonium citrate 0.5g/L, yeast extract 5g/L, fish peptone 10g/L, corn steep liquor 20g/L, magnesium sulfate heptahydrate 0.5g/L, L-methionine 2g/L, L-cysteine 2g/L, VB 1.005 g/L, VB 6.005 g/L, VB 12.005 g/L, saline solution 1ml/L, isopropyl-beta-D-thiogalactoside 48mg/L;

feed medium: 700g/L of glucose, 30g/L of methyl histidine, 5g/L of betaine hydrochloride, 2g/L of monopotassium phosphate, 5g/L of ammonium sulfate, 5g/L of yeast extract, 10g/L of fish peptone, 20g/L of corn steep liquor, 100g/L of glycerin, 0.01g/L of VB12, 30g/L of L-methionine, 30g/L of L-cysteine and 1ml/L of saline solution.

After 72 hours of fermentation, the OD600 is 121.2, and compared with example 1, the ergothioneine content is improved by 435%.

Example 8

Fermenting in a fermentation tank: the inoculation amount is 5 percent, the fermentation volume is 2L/5L, the initial rotating speed is 400rpm, the initial ventilation amount is 120L/h, the culture temperature is 37 for 0h-12h, the culture temperature is 30 for 12h-24h, the culture temperature is 28 for 24h-48h, the culture temperature is 25 for 48h-72h, the pH is controlled to 7.0, the dissolved oxygen is maintained to 20-30 percent, the dissolved oxygen is higher than 50 percent, the feeding is started, and the feeding and the dissolved oxygen combined control are performed.

Fermentation medium: glucose 20g/L, methyl histidine 2g/L, betaine hydrochloride 5g/L, monopotassium phosphate 0.5g/L, dipotassium phosphate 2g/L, ammonium sulfate 5g/L, diammonium citrate 0.5g/L, ferric ammonium citrate 0.5g/L, yeast extract 5g/L, fish peptone 10g/L, corn steep liquor 20g/L, magnesium sulfate heptahydrate 0.5g/L, L-histidine 2g/L, L-methionine 2g/L, L-cysteine 2g/L, VB 1.005 g/L, VB 6.005 g/L, VB 12.005 g/L, saline solution 1ml/L, isopropyl-beta-D-thiogalactoside 48mg/L, and water as a solvent;

feed medium: 700g/L of glucose, 30g/L of methyl histidine, 5g/L of betaine hydrochloride, 2g/L of monopotassium phosphate, 5g/L of ammonium sulfate, 5g/L of yeast extract, 10g/L of fish peptone, 20g/L of corn steep liquor, 100g/L of glycerin, 0.01g/L of VB12, 10g/L of L-histidine, 30g/L of L-methionine, 30g/L of L-cysteine and 1ml/L of salt solution.

At the end of 72h fermentation, the OD600 was 110.5, which increased the ergothioneine content by 554% compared to example 1.

The data of examples 1, 5-8 are compared and the results are shown in Table 1, where example 1 is the initial process and examples 5-8 are the modified processes.

Table 1: comparison of 5L fermenter example data

Example 5 demonstrates that increasing the organic nitrogen source in culture can increase biomass and also benefit the yield of ergothioneine.

Example 6 demonstrates that increasing histidine in culture, increasing substrate for ergothioneine synthesis, is also beneficial to ergothioneine yield.

Example 7 the use of methylhistidine instead of L-histidine on the basis of example 6 significantly increased ergothioneine yield by 435% compared to example 1.

Example 8 increased histidine on the basis of example 7, and the ergothioneine yield continued to increase, with 554% increase in ergothioneine yield and up to 7.2g/L compared to example 1.

Claims

1. The application of methyl histidine in ergothioneine production bacteria fermentation medium.

2. Use according to claim 1, wherein the ergothioneine-producing strain is a microorganism catalyzing the synthesis of ergothioneine from L-histidine, in particular a microorganism catalyzing the synthesis of ergothioneine from histidine, methionine and cysteine, preferably an engineered escherichia coli strain, more preferably an engineered escherichia coli strain as reported in patent document cn202310214002.X.

3. The use according to claim 1 or 2, wherein the methylhistidine is used in an amount of 0.5-10g/L in the ergothioneine-producing fermentation medium.

4. A fermentation medium of ergothioneine production bacteria is characterized in that 0.5-10g/L methyl histidine is added into a common fermentation medium of ergothioneine production bacteria.

5. The ergothioneine-producing strain fermentation medium of claim 4, wherein the common fermentation medium comprises: 10g/L to 40g/L of glucose, 2g/L to 20g/L of peptone, 2g/L to 40g/L of yeast extract, 10g/L to 40g/L of corn steep liquor, 0.1 g/L to 1g/L of magnesium sulfate heptahydrate and 7g/L to 40g/L of buffer salt, wherein the buffer salt comprises the following components: 1-10g/L of betaine hydrochloride, 0.2-1g/L of monopotassium phosphate, 0.5-5g/L of dipotassium phosphate, 5-30g/L of ammonium sulfate, 0.2-1g/L of diammonium hydrogen citrate and 0.1-1.0g/L of ferric ammonium citrate.

6. The ergothioneine-producing strain fermentation medium of claim 5, further comprising 1.5-10g/L of an ergothioneine-synthesizing precursor amino acid, the precursor amino acid comprising the following: 0.5-5g/L, L-methionine 0.5-5 g/L-histidine and L-cysteine 0.5-5g/L; and/or

0.003-0.03g/L of vitamins, wherein the vitamins comprise the following components: VB 1.001-0.01 g/L, VB 60.001.001-0.01 g/L and VB120.001-0.01g/L.

7. The ergothioneine-producing strain fermentation medium of claim 5, further comprising 30-200mg/L isopropyl-beta-D-thiogalactoside.

8. The ergothioneine-producing strain fermentation medium of claim 5, further comprising a salt solution of 0.2-2ml/L, the salt solution having a composition of: feSO ₄ ·7H ₂ O 10g/L，CuCl ₂ 0.2g/L，MnSO ₄ ·5H ₂ O 1g/L，CaCl ₂ ·2H ₂ O 0.2g/L，(NH ₄ ) ₆ Mo ₇ O ₂₄ 2g/L。

9. Use of a fermentation medium of an ergothioneine-producing strain according to any of claims 4-8 in the fermentative production of ergothioneine.

10. Use according to claim 9, characterized in thatThe fermentation further comprises a feed medium, wherein the feed medium comprises the following components: 700g/L of glucose, 30g/L of methyl histidine, 5g/L of betaine hydrochloride, 2g/L of monopotassium phosphate, 10g/L of ammonium sulfate, 5g/L of yeast extract, 5g/L of peptone, 20g/L of corn steep liquor, 100g/L of glycerin, 0.01g/L of VB12, 10g/L of L-histidine, 30g/L of L-methionine and 30g/L of L-cysteine, and 1ml/L of saline solution, wherein the composition of the saline solution is as follows: feSO ₄ ·7H ₂ O 10g/L，CuCl ₂ 0.2g/L，MnSO ₄ ·5H ₂ O 1g/L，CaCl ₂ ·2H ₂ O 0.2g/L，(NH ₄ ) ₆ Mo ₇ O ₂₄ 2g/L。