Expression cassette, genetically engineered bacterium for producing ergothioneine and application of genetically engineered bacterium

Abstract

The invention discloses an expression cassette, a genetically engineered bacterium for producing ergothioneine and application of the genetically engineered bacterium. The expression cassette comprises coding genes of Ncegt1, egtEncr and egtDAfr, and the coding genes of the egtEncr and the egtDAfr are shown in the description. The amino acid sequences of Ncegt1, egtEncr and egtDAfr are respectively as shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. The expression cassette has the advantage of being capable of improving the fermentation yield of the ergothioneine, and the genetically engineered bacterium for producing the ergothioneine can autonomously produce histidine which is one of substrates, has the advantages of being high in fermentation yield, high in production intensity and low in cost when being applied to production, and has industrial application prospects.

Description

Technical Field

[0001] This invention belongs to the field of synthetic biology, specifically relating to an expression cassette, a genetically engineered bacterium for producing ergothionein, and their applications. Background Technology

[0002] Ergothioneine (ERG / EGT) is a thiol-containing histidine betaine derivative with a relatively high reduction potential (-60 mV) and natural and potent antioxidant activity. This makes it more stable and less prone to auto-oxidation compared to other thiol-containing antioxidants (such as glutathione). EGT has functions such as relieving inflammation, preventing radiation damage, protecting cellular physiological functions, and anti-aging. Therefore, it shows broad application prospects in the fields of medicine, cosmetics, and functional foods.

[0003] The main methods for preparing EGT include three types: natural product extraction, chemical synthesis, and biosynthesis.

[0004] EGT naturally accumulates in certain edible fungi, cyanobacteria, actinomycetes, and methylbacteria. Within a species, it is synthesized through specific metabolic pathways. The biggest problem facing natural product extraction methods is the low accumulation of products, the high cost of fermentation and extraction processes, and the lack of possibility for industrial production.

[0005] The chemical synthesis method uses histidine as the starting material and involves multiple steps such as thiolation, thioprotection, methylation, and deprotection. The route is redundant and the yield is very low.

[0006] Obtaining EGT through biosynthesis offers advantages such as readily available raw materials, low cost, mild reaction conditions, and high yield. Currently, several industrial strains can produce EGT, with *E. coli* being the most common host. Chen [1] Simultaneous expression of Trichoderma reesei in Escherichia coli Two ERG biosynthetic genes After 48 hours of shake-flask culture, the EGT yield reached 70.59 mg / L; through fed-batch fermentation, after 143 hours of culture in a 2-liter fermenter, the extracellular ERG yield reached 4.34 g / L. Biosynthesis is currently the most promising method for EGT production, but all current biosynthetic methods require the exogenous addition of L-methionine, L-cysteine, and L-histidine. These three precursor amino acids are expensive, batch-to-batch quality variations can lead to fluctuating results, and exogenous addition requires separate sterilization, batch addition, and precise control of feeding time and concentration; otherwise, cell growth may be inhibited or waste may occur, significantly increasing the difficulty of industrial production and raw material costs. Furthermore, existing technologies also suffer from drawbacks such as long culture cycles, low production intensity, and low fermentation yields.

[0007] References:

[0008] [1] Chen, Z., He, Y., Wu, X., Wang, L., Dong, Z., & Chen, X. Toward more efficient ergothioneine production using the fungal ergothioneinebiosynthetic pathway[J]. Microbial Cell Factories, 2022, 21(1), 76. Summary of the Invention

[0009] To address the shortcomings of existing methods for producing ergothioneine, which require the exogenous addition of histidine and suffer from high costs, low production intensity, and low fermentation yield, this invention provides an expression cassette, a genetically engineered bacterium for ergothioneine production, and their applications. The expression cassette provided by this invention has the advantage of increasing ergothioneine fermentation yield, and the genetically engineered bacterium for ergothioneine production can autonomously produce histidine, one of the substrates. When applied to production, it offers advantages such as reduced costs, high fermentation yield, and high production intensity, showing promise for industrial application.

[0010] The present invention solves the above-mentioned technical problems through the following technical solutions:

[0011] The first aspect of the present invention provides an expression cassette comprising the coding genes for Ncegt1, egtEncr, and egtDAfr; the amino acid sequences of Ncegt1, egtEncr, and egtDAfr are shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively.

[0012] In some embodiments of the present invention, the expression cassette contains, from 5' to 3', the coding gene for Ncegt1, the coding gene for egtDAfr, and the coding gene for egtEncr.

[0013] In some embodiments of the present invention, the expression cassette contains, from 5' to 3', the coding gene for egtDAfr, the coding gene for Ncegt1, and the coding gene for egtEncr.

[0014] In some embodiments of the present invention, the expression cassette further includes a promoter, an enhancer, a terminator, and / or a ribosome binding site.

[0015] In some preferred embodiments of the present invention, the promoter is a strongly inductive promoter.

[0016] In some specific embodiments of the present invention, the promoter is a trc promoter.

[0017] In some embodiments of the present invention, the coding genes for Ncegt1, egtEncr, and egtDAfr respectively have nucleotide sequences as shown in SEQ ID NO: 13-15.

[0018] A second aspect of the present invention provides a recombinant expression vector comprising an expression cassette as described in the first aspect.

[0019] In some embodiments of the present invention, the copy number of the recombinant expression vector is 5-20 copies / cell.

[0020] In some embodiments of the present invention, the backbone plasmid of the recombinant expression vector is pACYCDuet-1.

[0021] A third aspect of the present invention provides a substrate bacteria for producing histidine, wherein the substrate bacteria satisfy at least one of the following requirements:

[0022] (1) The expression of the following genes in the *Bacteroides truncatum* is reduced or lost: the gene encoding the histidine operon leader sequence hisL and the gene encoding the purine repressor protein purR; or,

[0023] The gene encoding the histidine operon leader sequence hisL, the gene encoding the purine repressor protein purR, and the gene encoding the bifunctional enzyme ushA with 5'-nucleotidase / UDP-glycolytic activity.

[0024] (2) The following genes are overexpressed in the *Bacteroides spp.*: the gene encoding a variant of ATP-phosphoribotransferase hisG, the gene encoding a variant of 5-phosphoribose-1-pyrophosphate synthase prsA, and the gene encoding glucose-6-phosphate dehydrogenase zwf; or,

[0025] The coding genes for variants of ATP-phosphoribotransferase hisG, variants of 5-phosphoribose-1-pyrophosphate synthase prsA, glucose-6-phosphate dehydrogenase zwf, bifunctional purine biosynthesis protein purH, and leucine efflux protein leuE.

[0026] The variant of ATP-phosphoribotransferase hisG, the variant of 5-phosphoribose-1-pyrophosphate synthase prsA, the glucose-6-phosphate dehydrogenase zwf, the bifunctional purine biosynthesis protein purH, and the leucine efflux protein leuE each contain the amino acid sequences shown in SEQ ID NO: 8-12.

[0027] In some embodiments of the present invention, the gene encoding the variant of the ATP-phosphoribotransferase hisG, the gene encoding the variant of the 5-phosphoribose-1-pyrophosphate synthase prsA, the gene encoding the glucose-6-phosphate dehydrogenase zwf, the gene encoding the bifunctional purine biosynthesis protein purH, and the gene encoding the leucine efflux protein leuE are overexpressed at sites hisG, yeep, yjip, mbhA, and mbhA, respectively.

[0028] In some specific embodiments of the present invention, the genotypes of the basal bacteria are ΔhisL, hiG::pTrc-hisG*, ΔpurR, yeep::pTrc-prsA*, yjip::pTrc-zwf, mbhA::pTrc-purH, ΔushA, yagH::pTrc-leuE.

[0029] In some preferred embodiments of the present invention, the reduction or loss of gene expression in the sclerotium is achieved by knockout.

[0030] In some embodiments of the present invention, the starting strain of the chassis bacteria is Escherichia coli w3110.

[0031] A fourth aspect of the present invention provides a genetically engineered bacterium for producing ergothioneine, the genetically engineered bacterium comprising an expression cassette as described in the first aspect or a recombinant expression vector as described in the second aspect.

[0032] In some embodiments of the present invention, the substrate bacteria of the genetically engineered bacteria are the substrate bacteria as described in the third aspect.

[0033] The fifth aspect of the present invention provides a method for preparing ergothioneine, the method comprising fermenting and culturing genetically engineered bacteria as described in the fourth aspect in a fermentation medium to obtain ergothioneine.

[0034] In some embodiments of the present invention, each liter of the fermentation medium comprises: 18-30 g of glucose, 4-8 g of ammonium sulfate, 1-5 g of sodium chloride, 1-5 g of potassium dihydrogen phosphate, 0.2-1 g of anhydrous magnesium sulfate, 0.1-0.5 g of ferric ammonium citrate, 10-15 g of yeast extract, 1-5 g of lysine, 0.1-0.8 g of calcium chloride, and 0.01-0.05 g of zinc sulfate heptahydrate.

[0035] In some preferred embodiments of the present invention, each liter of the fermentation medium comprises: 20 g glucose, 5 g ammonium sulfate, 2 g sodium chloride, 2 g potassium dihydrogen phosphate, 0.5 g anhydrous magnesium sulfate, 0.1 g ferric ammonium citrate, 12 g yeast extract, 2 g lysine, 0.105 g calcium chloride and 0.01 g zinc sulfate heptahydrate.

[0036] In some embodiments of the present invention, each liter of the fermentation medium further comprises: 2-3 mg of zinc sulfate heptahydrate, 1-2 mg of anhydrous calcium chloride, 1.5-2.5 mg of sodium molybdate dihydrate, 1-2 mg of copper sulfate pentahydrate, 0.4-0.6 mg of boric acid and 0.05-0.2 mL of hydrochloric acid.

[0037] In some preferred embodiments of the present invention, each liter of the fermentation medium further comprises: 2.76 mg zinc sulfate heptahydrate, 1.014 mg anhydrous calcium chloride, 2 mg sodium molybdate dihydrate, 1.9 mg copper sulfate pentahydrate, 0.5 mg boric acid, and 0.1 mL hydrochloric acid.

[0038] In some embodiments of the present invention, each liter of the fermentation medium comprises: 18-30 g glucose, 4-8 g ammonium sulfate, 1-5 g sodium chloride, 1-5 g potassium dihydrogen phosphate, 0.2-1 g anhydrous magnesium sulfate, 0.1-0.5 g ferric ammonium citrate, 10-15 g yeast extract, 1-5 g lysine, 0.1-0.8 g calcium chloride, 0.01-0.05 g zinc sulfate heptahydrate, 2-3 mg zinc sulfate heptahydrate, 1-2 mg anhydrous calcium chloride, 1.5-2.5 mg sodium molybdate dihydrate, 1-2 mg copper sulfate pentahydrate, 0.4-0.6 mg boric acid, and 0.05-0.2 mL hydrochloric acid.

[0039] In some specific embodiments of the present invention, each liter of the fermentation medium comprises: 20 g glucose, 5 g ammonium sulfate, 2 g sodium chloride, 2 g potassium dihydrogen phosphate, 0.5 g anhydrous magnesium sulfate, 0.1 g ferric ammonium citrate, 12 g yeast extract, 2 g lysine, 0.105 g calcium chloride, 0.01 g zinc sulfate heptahydrate, 1 g L-cysteine, 1.014 mg anhydrous calcium chloride, 2 mg sodium molybdate dihydrate, 1.9 mg copper sulfate pentahydrate, 0.5 mg boric acid, and 0.1 mL hydrochloric acid.

[0040] In some embodiments of the present invention, the fermentation culture includes steps selected from the following:

[0041] (i) Inoculation: Inoculate the genetically engineered bacteria as described in the third aspect into liquid culture medium and incubate at 25-37°C and / or 180-300 rpm for 6-15 h;

[0042] (ii) Pre-culture: The culture obtained in (i) is transferred to the fermentation medium for pre-culture; the inoculum size is 1-5%, and / or the culture temperature is 25-37°C, and / or the shaking speed is 180-300 rpm, until OD. 600 The value is 0.9-1.2, for example, 1;

[0043] (iii) Induction of expression: Add IPTG, L-methionine, and L-cysteine; preferably, add IPTG at a final concentration of 0.1-0.2 mM and / or L-methionine and L-cysteine ​​at final concentrations of 0.5-3 g / L, and / or continue culturing at an induction temperature of 25-37℃ and / or 220 rpm for 25-50 h; and / or,

[0044] (iv) Product collection.

[0045] In some embodiments of the present invention, the conditions for inducing expression during the fermentation culture include: a temperature of 25-37°C, such as 25-35, 25-33, 30-33, 30-35, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35°C; and / or an oscillation speed of 180-300 rpm, such as 180-260, 200-240, 210-220, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 rpm; and / or a pH of 6-8, such as 6.5-7.5, 6.8-7.2, 6.5, 7, or 7.5.

[0046] In some embodiments of the present invention, the induction time for expression in the fermentation culture is 25-50 h, for example, 30-50, 35-50, 40-50, 45-50, 30, 35, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 h.

[0047] In some embodiments of the present invention, during the induction of expression in the fermentation culture, IPTG at a final concentration of 0.1-0.2 mM and / or L-methionine and L-cysteine ​​at final concentrations of 0.5-3 g / L are added.

[0048] In some specific embodiments of the present invention, the final concentration of IPTG is 0.1, 0.15, or 0.2 mM.

[0049] In some specific embodiments of the present invention, the final concentrations of L-methionine and L-cysteine ​​are 0.5, 1, 1.5, 2, 2.5 or 3 g / L, respectively.

[0050] The sixth aspect of the present invention provides a method for preparing genetically engineered bacteria that produce ergothioneine, the method comprising constructing an expression cassette as described in the first aspect, a recombinant expression vector as described in the second aspect, and / or a histidine-producing chassis bacterium as described in the third aspect.

[0051] In some embodiments of the present invention, the method further includes the step of introducing an expression cassette as described in the first aspect or a recombinant expression vector as described in the second aspect into a histidine-producing spore as described in the third aspect.

[0052] In this invention, the term "starting strain" refers to a commercially available original model strain that has been genetically modified without the methods disclosed in this invention, retaining its genotype and phenotype, and is the genetic starting point for the metabolic engineering modification of the strains in this invention.

[0053] In this invention, the terms "chassis bacteria" or "chassis strain" are synonymous, referring to a host strain with stable production performance and a clear genetic background obtained through genetic modification (e.g., deleting competing pathways, optimizing regulatory networks, introducing exogenous modules, etc.) based on the "starting strain," which can serve as a platform for the subsequent synthesis of end products. In this invention, "chassis bacteria" or "chassis strain" can also refer to a genetically modified strain that produces high levels of histidine.

[0054] In this invention, the term "expression cassette" refers to a DNA unit modularly assembled in a "5'-3' direction" that can directly drive the transcription and translation of the target gene. It possesses the following functional characteristics: functional integrity, enabling expression in various heterologous hosts; portability: it can be "plug-and-play" between different plasmids, viral vectors, or specific genomic sites via methods such as enzyme digestion, Gibson, and Golden Gat; regulatory: its promoter, RBS, enhancer, terminator, and degradation tag are replaceable, allowing for precise control over expression intensity, induction mode, and cellular localization; and stackability: multiple expression cassettes can be tandemly linked into multi-gene modules for one-time introduction into pathways or synthetic routes.

[0055] In this invention, the term "coding gene" or "gene" refers to a polynucleotide sequence that can be transcribed into mRNA and subsequently translated into a functional polypeptide / protein having a specific amino acid sequence. These two terms may be used interchangeably in this invention. In some embodiments, the coding sequence is a complementary DNA (cDNA) sequence reverse transcribed from messenger RNA (mRNA). In some embodiments, the coding sequence is mRNA.

[0056] In this invention, the term "overexpression" refers to the genetic manipulation that causes the transcription or translation level of a target gene in a host cell to be significantly higher than its natural state.

[0057] The disadvantages of existing technology are:

[0058] 1. The biggest problem with natural product extraction methods is the low product accumulation rate, high fermentation and extraction process costs, and the lack of possibility for industrial production.

[0059] 2. The chemical synthesis of EGT faces several challenges, including the difficulty in preparing the raw material 2-mercaptoimidazole, the difficulty in post-processing the product, and the difficulty in obtaining high-purity EGT.

[0060] 3. Biosynthesis is currently the most promising method for EGT production. However, it requires the exogenous addition of L-methionine, L-cysteine, and L-histidine, which significantly increases the difficulty of industrial production and raw material costs, resulting in problems such as high cost, long cultivation cycle, low production intensity, and low fermentation yield.

[0061] The technical problem solved by this invention is to provide a low-cost genetically engineered bacterium for synthesizing EGT, which can autonomously produce histidine, one of the substrates, and then achieve high EGT production in shake flasks with zero added histidine by combining different expression cassettes (containing key genes for EGT synthesis).

[0062] The purpose of this invention is to construct a high-yield EGT engineered strain, which uses high-histidine-producing Escherichia coli as the host and heterologously expresses genes related to the EGT synthesis pathway to achieve high EGT production without the need for histidine supply.

[0063] Specifically, key synthetic pathways for EGT production were constructed by selecting Tregt1 and Tregt2 from Trichoderma reesei, truncated Ncegt1 and egtEncr from Neurospora crassa, egtDAfr from Microcoleus sp. PCC7113, and MsegtD and MsegtE from Mycobacterium smegmatis. Multiple combined pathways were developed to produce EGT, and the optimal combination was determined. High-histidine-producing chassis strains were obtained through metabolic engineering of E. coli. The EGT yield of the optimal combination in the high-histidine-producing chassis strain and the ordinary strain were compared to determine the yield and cost advantages of the high-histidine-producing strain. Shake-flask culture conditions, such as induction temperature, were optimized.

[0064] The technical solution is explained in detail in four points:

[0065] 1. Construction and Expression of EGT Synthesis Pathway

[0066] Based on the literature, the inventors selected Tregt1 and Tregt2 from Trichoderma reesei, truncated Ncegt1 and egtEncr from Neurospora crassa, egtDAfr from Microcoleus sp. PCC 7113, and from Mycobacterium MsegtD and MsegtE of smegmatis were expressed on the pACYCDuet-1 vector, and the expression of Tregt1, Tregt2, Ncegt1, egtEncr, egtDAfr, MsegtD, and MsegtE was determined. Subsequently, different combinations, such as Ncegt1-egtDAfr-egtEncr, egtDAfr-Ncegt1-egtEncr, Tregt1-egtDAfr-egtEncr, Tregt1-egtEncr, Ncegt1-Tregt1-egtEncr, Tregt1-Ncegt1-egtEncr, Tregt2-Tregt1, and Ncegt1-MsegtD-MsegtE, were expressed on the pACYCDuet-1 vector.

[0067] 2. Histidine chassis strains were used to verify the yield of different combinations.

[0068] In high-histidine-producing chassis strains, the advantages and disadvantages of different EGT production routes were verified by shaking flask fermentation without the addition of histidine to the culture medium.

[0069] 3. Compare the yields of BL21(DE3) and histidine discs.

[0070] The optimal combination was constructed into the BL21(DE3) strain and cultured in shake flasks with 1 g / L of histidine, methionine, and cysteine ​​added to the culture medium. The EGT production of BL21(DE3) and a high-histidine-producing chassis strain were compared.

[0071] 4. Optimize shake flask culture conditions to increase shake flask yield.

[0072] The induction temperature and other parameters of the strain were optimized during shake-flask fermentation, and the EGT yield under different conditions was compared.

[0073] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0074] The reagents and raw materials used in this invention are all commercially available.

[0075] The positive and progressive effects of this invention are as follows: the expression cassette provided by this invention has the advantage of increasing the fermentation yield of ergothioneine, and the genetically engineered bacteria that produce ergothioneine can autonomously produce histidine, one of the substrates. When applied to production, it has the advantages of high fermentation yield, high production intensity and reduced cost, and has the prospect of industrial application. Attached Figure Description

[0076] Figure 1 To verify different combinations of EGT synthesis methods in shake-flask fermentation culture of high-histidine-producing chassis strains.

[0077] Figure 2 To verify the Ncegt1-egtDAfr-egtEncr combination in shake flasks using the BL21(DE3) chassis strain.

[0078] Figure 3 To investigate the effect of induction temperature on EGT synthesis by strain EGT1.

[0079] Figure 4 The ability of high-amino acid-producing chassis strains to produce histidine. Detailed Implementation

[0080] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0081] Example 1: Construction and Expression of EGT Synthesis Pathway

[0082] To construct the synthetic pathway of EGT, the inventors selected Tregt1 and Tregt2 from Trichoderma reesei, truncated Ncegt1 and egtEncr from Neurospora crassa, egtDAfr from Microcoleus sp. PCC 7113, and MsegtD and MsegtE from Mycobacterium smegmatis, based on literature. The amino acid sequences are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, respectively, as shown in Table 1. Tregt1, Tregt2, Ncegt1, egtEncr, egtDAfr, MsegtD, and MsegtE were expressed on the pACYCDuet-1 vector. It was determined that Tregt1, Ncegt1, egtEncr, egtDAfr, MsegtD, and MsegtE had relatively good expression levels, while Tregt2 had a relatively low expression level.

[0083] Subsequently, through different combinations, specifically Ncegt1-egtDAfr-egtEncr, egtDAfr-Ncegt1-egtEncr, Tregt1-egtDAfr-egtEncr, Tregt1-egtEncr, Ncegt1-Tregt1-egtEncr, Tregt1-Ncegt1-egtEncr, Tregt2-Tregt1, Ncegt1-MsegtD-MsegtE, etc., expression cassettes of trc promoter-A gene-RBS site-B gene-trc promoter-C gene or trc promoter-A gene-trc promoter-B gene were used, and expressed on the pACYCDuet-1 vector. The sequence of the RBS site is ATAGGAGAT.

[0084] Example 2: Validation of yield of different combinations in high-histidine-producing chassis strains

[0085] 1. Construction of high-histidine-producing chassis strains

[0086] A high-histidine-producing chassis strain of Escherichia coli was constructed using CRISPR / Cas9 technology according to the literature (Acta Biochim Biophys Sin, 2021, 53(5), 620–627), with the starting strain being W3110.

[0087] In the starting strain W3110, the histidine operon leader sequence hisL was knocked out to remove histidine attenuation regulation. Based on this, hiG was replaced with pTrc-hisG* from *E. coli* to remove histidine-mediated feedback inhibition; the histidine G* sequence is SEQ ID NO: 8. Further knockout of the purR gene removed global repression of purine synthesis genes, enhancing the accumulation of precursor PRPP. Further integration of anti-feedback pTrc-prsA* from *E. coli* at the non-essential gene yeep site enhanced precursor PRPP accumulation; the prsA* sequence is SEQ ID NO: 9. Further overexpression of pTrc-zwf from *E. coli* at the non-essential gene yjip site enhanced the metabolic flux of the pentose phosphate pathway; the zwf sequence is SEQ ID NO: 10. Further overexpression of pTrc-purH from *E. coli* at the non-essential gene mbhA site strengthened the purine synthesis pathway; the purH sequence is SEQ ID NO: 10. 11; Further knockout of the ushA gene reduced IMP degradation and enhanced the supply of precursor ATP; further, overexpression of pTrc-leuE from E. coli at the non-essential gene yagH site enhanced histidine efflux. The sequence of leuE is SEQ ID NO: 12. The final high-hitidine-producing chassis strain (HIS1) was obtained: E. coli W3110, ΔhisL, hiG::pTrc-hisG*, ΔpurR, yeep::pTrc-prsA*, yjip::pTrc-zwf, mbhA::pTrc-purH, ΔushA, yagH::pTrc-leuE.

[0088] To investigate the histidine production capacity of the high-yield histidine chassis strain, the strain was subjected to shake-flask fermentation. The results are shown below. Figure 4 The results showed that after 48 hours of shake-flask culture, the high-histidine-producing chassis strain was able to produce 1.673 g / L of histidine.

[0089] The specific steps for shake-flask fermentation are as follows:

[0090] The shake-flask fermentation medium for histidine production using *Bacillus subtilis* consists of: glucose 20 g / L, ammonium sulfate 5 g / L, sodium chloride 2 g / L, potassium dihydrogen phosphate 2 g / L, anhydrous magnesium sulfate 0.5 g / L, ferric ammonium citrate 0.1 g / L, yeast extract 12 g / L, lysine 2 g / L, calcium chloride 0.105 g / L, zinc sulfate heptahydrate 0.01 g / L, and trace elements 1 mL / L. The pH is adjusted to 7 with ammonia.

[0091] Trace elements (1 L): 2.76 g zinc sulfate heptahydrate, 1.014 g anhydrous calcium chloride, 2.0 g sodium molybdate dihydrate, 1.9 g copper sulfate pentahydrate, 0.5 g boric acid, 100 mL hydrochloric acid.

[0092] Shake-flask fermentation method: Inoculate single colonies from the plate into 5 mL of LB broth containing the appropriate antibiotic and incubate overnight at 37°C and 220 rpm. Transfer the culture at a 2% inoculation rate to 25 mL (250 mL baffled Erlenmeyer flask) of shake-flask fermentation medium. Incubate at 37°C for 3-4 hours until OD (discharge rate) reaches 100%. 600 Add IPTG to a final concentration of 0.1 mM and continue culturing at an induction temperature of 33℃ and 220 rpm for 45 h. Collect the fermentation broth, centrifuge, and take the supernatant to detect histidine.

[0093] 2. Construction of genetically engineered bacteria for high ergothioneine production

[0094] To verify the yield of different combinations in high-histidine-producing chassis strains, the different EGT synthesis combinations described in Example 1—Ncegt1-egtDAfr-egtEncr, egtDAfr-Ncegt1-egtEncr, Tregt1-egtDAfr-egtEncr, Tregt1-egtEncr, Ncegt1-Tregt1-egtEncr, Tregt1-Ncegt1-egtEncr, Tregt2-Tregt1, and Ncegt1-MsegtD-MsegtE—were transformed into high-histidine-producing chassis strains, yielding strains EGT1 to EGT8. The superiority of different EGT production methods for strains EGT1 to EGT8 was verified through shake-flask fermentation without the addition of histidine to the culture medium. Results are shown below. Figure 1 As shown, the results indicate that the optimal EGT route combination is Ncegt1-egtDAfr-egtEncr (strain EGT1) or egtDAfr-Ncegt1-egtEncr (strain EGT2), with EGT yields reaching 323 mg / L or 302 mg / L respectively.

[0095] The specific steps for shake-flask fermentation are as follows:

[0096] Shake-flask fermentation medium: glucose 20 g / L, ammonium sulfate 5 g / L, sodium chloride 2 g / L, potassium dihydrogen phosphate 2 g / L, anhydrous magnesium sulfate 0.5 g / L, ferric ammonium citrate 0.1 g / L, yeast extract 12 g / L, lysine 2 g / L, calcium chloride 0.105 g / L, zinc sulfate heptahydrate 0.01 g / L, and trace elements 1 mL / L. The pH was adjusted to 7 with ammonia.

[0097] Trace elements (1L): 2.76 g zinc sulfate heptahydrate, 1.014 g anhydrous calcium chloride, 2.0 g sodium molybdate dihydrate, 1.9 g copper sulfate pentahydrate, 0.5 g boric acid, 100 mL hydrochloric acid.

[0098] Shake-flask fermentation method: Inoculate single colonies from the plate into 5 mL of LB broth containing the appropriate antibiotic and incubate overnight at 37°C and 220 rpm. Transfer the culture at a 2% inoculation rate to 25 mL (250 mL baffled Erlenmeyer flask) of shake-flask fermentation medium. Incubate at 37°C for 3-4 hours until OD (discharge rate) reaches 100%. 600 Add IPTG to a final concentration of 0.1 mM and L-methionine and L-cysteine ​​to a final concentration of 1 g / L, and continue culturing at an induction temperature of 33℃ and 220 rpm for 45 h. Collect the fermentation broth, centrifuge, and take the supernatant to detect EGT.

[0099] Example 3: Comparison of EGT production of the optimal combination Ncegt1-egtDAfr-egtEncr in BL21(DE3) and high-histidine-producing chassis strains

[0100] To investigate the ability of the optimal combination to produce EGT in other chassis strains, the inventors compared chassis strain BL21(DE3) with high-histidine-producing chassis strains. The optimal combination Ncegt1-egtDAfr-egtEncr was constructed into the pACYCDuet-1 and pETDuet-1 vectors according to the expression cassette of trc promoter-A gene-RBS site-B gene-trc promoter-C gene, and transformed into BL21(DE3) strain, yielding strains EGT9 and EGT10, respectively.

[0101] Strains EGT9, EGT10, and the control strain EGT1 were subjected to shake-flask fermentation, and the results are as follows: Figure 2 As shown, the results indicated that the optimal combination Ncegt1-egtDAfr-egtEncr synthesized EGT concentrations of 24 mg / L and 61 mg / L on pACYCDuet-1 (strain EGT9) and pETDuet-1 (strain EGT10) vectors, respectively. This suggests that the optimal combination Ncegt1-egtDAfr-egtEncr produced different EGT concentrations during shake-flask fermentation when expressed on plasmid vectors with different copy numbers; higher copy numbers resulted in higher EGT yields. However, compared to high-histidine-producing chassis strains, the EGT yield of the Ncegt1-egtDAfr-egtEncr combination in high-histidine-producing chassis strains was 5.5 times that of BL21(DE3), and no additional histidine was required in the culture medium, reducing fermentation costs.

[0102] The specific steps for shake-flask fermentation are as follows:

[0103] Shake-flask fermentation medium: glucose 20 g / L, ammonium sulfate 5 g / L, sodium chloride 2 g / L, potassium dihydrogen phosphate 2 g / L, anhydrous magnesium sulfate 0.5 g / L, ferric ammonium citrate 0.1 g / L, yeast extract 12 g / L, lysine 2 g / L, calcium chloride 0.105 g / L, zinc sulfate heptahydrate 0.01 g / L, and trace elements 1 mL / L. The pH was adjusted to 7 with ammonia.

[0104] Trace elements (1 L): 2.76 g zinc sulfate heptahydrate, 1.014 g anhydrous calcium chloride, 2.0 g sodium molybdate dihydrate, 1.9 g copper sulfate pentahydrate, 0.5 g boric acid, 100 mL hydrochloric acid.

[0105] Shake-flask fermentation method: Inoculate single colonies from the plate into 5 mL of LB broth containing the appropriate antibiotic and incubate overnight at 37°C and 220 rpm. Transfer the culture at a 2% inoculation rate to 25 mL (250 mL baffled Erlenmeyer flask) of shake-flask fermentation medium. Incubate at 37°C for 3-4 hours until OD (discharge rate) reaches 100%. 600 Add IPTG to a final concentration of 0.1 mM, and L-methionine and L-cysteine ​​to a final concentration of 1 g / L (for fermentation strain EGT1), or L-methionine, L-cysteine, and L-histidine to a final concentration of 1 g / L (for fermentation strains EGT9 or EGT10), and continue culturing at an induction temperature of 33℃ and 220 rpm for 45 h. Collect the fermentation broth, centrifuge, and take the supernatant to detect EGT.

[0106] Example 4: Optimizing induction temperature to increase shake flask yield

[0107] To further improve EGT yield, the inventors optimized the induction temperature of strain EGT1 during shake-flask fermentation and compared the EGT yield under different induction temperature conditions. Strain EGT1 was subjected to shake-flask fermentation at 25℃, 30℃, 33℃, and 37℃, with the following results: Figure 3 As shown in the figure. The results indicated that a final OD600 was moderate at an induction temperature of 33℃, and strain EGT1 produced the highest EGT yield, with a concentration of 342 mg / L. This suggests that excessively high induction temperatures may not be suitable for the expression of key genes involved in EGT synthesis, while excessively low temperatures may negatively impact strain growth.

[0108] The specific steps for shake-flask fermentation are as follows:

[0109] Shake-flask fermentation medium: glucose 20 g / L, ammonium sulfate 5 g / L, sodium chloride 2 g / L, potassium dihydrogen phosphate 2 g / L, anhydrous magnesium sulfate 0.5 g / L, ferric ammonium citrate 0.1 g / L, yeast extract 12 g / L, lysine 2 g / L, calcium chloride 0.105 g / L, zinc sulfate heptahydrate 0.01 g / L, and trace elements 1 ml / L. The pH was adjusted to 7 with ammonia.

[0110] Trace elements (1 L): 2.76 g zinc sulfate heptahydrate, 1.014 g anhydrous calcium chloride, 2.0 g sodium molybdate dihydrate, 1.9 g copper sulfate pentahydrate, 0.5 g boric acid, 100 mL hydrochloric acid.

[0111] Shake-flask fermentation method: Inoculate single colonies from the plate into 5 mL of LB broth containing the appropriate antibiotic and incubate overnight at 37°C and 220 rpm. Transfer the culture at a 2% inoculation rate to 25 mL (250 mL baffled Erlenmeyer flask) of shake-flask fermentation medium. Incubate at 37°C for 3-4 hours until OD (discharge rate) reaches 100%. 600 Add IPTG to a final concentration of 0.1 mM and L-methionine and L-cysteine ​​to a final concentration of 1 g / L, and continue culturing at the induction temperature (25℃, 30℃, 33℃, or 37℃) and 220 rpm for 45 h. Collect the fermentation broth, centrifuge, and take the supernatant to detect EGT.

[0112] The sequence information involved in this invention is shown in Table 1.

[0113] Table 1 Sequence Information

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120] Although this disclosure has been specifically shown and described by way of examples, those skilled in the art will understand that various changes in form and detail may be made without departing from the scope of the disclosure.

Claims

1. An expression cassette comprising, The expression cassette comprises coding genes of Ncegt1, egtEncr and egtDAfr; the amino acid sequences of Ncegt1, egtEncr and egtDAfr are respectively shown as SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO:

5.

2. The expression cassette of claim 1, wherein, The expression cassette comprises, from 5' to 3': (1) coding genes of the Ncegt1, the egtDAfr and the egtEncr; or, (2) coding genes of the egtDAfr, the Ncegt1 and the egtEncr; And / or, the expression cassette further comprises a promoter, an enhancer, a terminator and / or a ribosome binding site; And / or, the coding genes of the Ncegt1, the egtEncr and the egtDAfr respectively have the nucleotide sequences shown as SEQ ID NO: 13-15.

3. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the expression cassette as claimed in claim 1 or 2.

4. A histidine-producing chassis, characterized by, The chassis fungus of the genetically engineered fungus meets at least one of the following requirements: (1) the expression of the following genes in the chassis fungus is reduced or lost: coding gene of histidine operon leader sequence hisL and coding gene of purine repressor purR; or, coding gene of histidine operon leader sequence hisL, coding gene of purine repressor purR and coding gene of bifunctional enzyme ushA with 5'-nucleotidase / UDP-sugar hydrolase activity; (2) the following genes are overexpressed in the chassis fungus: coding gene of variant of ATP-phosphoribosyltransferase hisG, coding gene of variant of 5-phosphoribosyl-1-pyrophosphate synthetase prsA and coding gene of glucose-6-phosphate dehydrogenase zwf; or, coding gene of variant of ATP-phosphoribosyltransferase hisG, coding gene of variant of 5-phosphoribosyl-1-pyrophosphate synthetase prsA, coding gene of glucose-6-phosphate dehydrogenase zwf, coding gene of bifunctional purine biosynthesis protein purH and coding gene of leucine efflux protein leuE; The variant of ATP-phosphoribosyltransferase hisG, the variant of 5-phosphoribosyl-1-pyrophosphate synthetase prsA, the glucose-6-phosphate dehydrogenase zwf, the bifunctional purine biosynthesis protein purH and the leucine efflux protein leuE respectively comprise the amino acid sequences shown as SEQ ID NO: 8-12.

5. The substrate fungus of claim 4, wherein, The starting strain of the ascomycete is Aspergillus niger w3110.

6. A genetically engineered bacteria for producing ergothioneine, characterized by, The genetically engineered fungus comprises the expression cassette as claimed in claim 1 or 2 or the recombinant expression vector as claimed in claim 3.

7. The genetically engineered bacteria as described in claim 6, characterized in that, The chassis fungus of the genetically engineered fungus is as claimed in claim 4 or 5.

8. A method of preparing ergothioneine, characterized by, The method comprises fermenting the genetically engineered fungus as claimed in claim 6 or 7 in a fermentation medium to obtain ergothioneine.

9. The method of claim 8, wherein, each liter of the fermentation medium comprises: glucose 18-30 g, ammonium sulfate 4-8 g, sodium chloride 1-5 g, potassium dihydrogen phosphate 1-5 g, anhydrous magnesium sulfate 0.2-1 g, ferric ammonium citrate 0.1-0.5 g, yeast extract 10-15 g, lysine 1-5 g, calcium chloride 0.1-0.8 g, and zinc sulfate heptahydrate 0.01-0.05 g; and / or, the conditions for inducing expression in the fermentation culture include: temperature of 25-37℃; and / or, shaking speed of 180-300 rpm; and / or, pH of 6-8; and / or, the time for inducing expression in the fermentation culture is 25-50 h; and / or, IPTG, L-methionine and L-cysteine are added during the induction of expression in the fermentation culture; preferably, IPTG with a final concentration of 0.1-0.2 mM and / or L-methionine and L-cysteine with a final concentration of 0.5-3 g / L, respectively, are added.

10. A method for preparing a genetically engineered bacteria for producing ergothioneine, characterized by, The method comprises constructing the expression cassette of claim 1 or 2, the recombinant expression vector of claim 3, and / or the chassis of claim 4 or 5; Preferably, the method further comprises the step of introducing the expression cassette of claim 1 or 2 or the recombinant expression vector of claim 3 into the chassis of claim 4 or 5. each liter of the fermentation medium comprises: glucose 18-30 g, ammonium sulfate 4-8 g, sodium chloride 1-5 g, potassium dihydrogen phosphate 1-5 g, anhydrous magnesium sulfate 0.2-1 g, ferric ammonium citrate 0.1-0.5 g, yeast extract 10-15 g, lysine 1-5 g, calcium chloride 0.1-0.8 g, and zinc sulfate heptahydrate 0.01-0.05 g; and / or, the conditions for inducing expression in the fermentation culture include: temperature of 25-37℃; and / or, shaking speed of 180-300 rpm; and / or, pH of 6-8; and / or, the time for inducing expression in the fermentation culture is 25-50 h; and / or, IPTG, L-methionine and L-cysteine are added during the induction of expression in the fermentation culture; preferably, IPTG with a final concentration of 0.1-0.2 mM and / or L-methionine and L-cysteine with a final concentration of 0.5-3 g / L, respectively, are added. The method comprises constructing the expression cassette of claim 1 or 2, the recombinant expression vector of claim 3, and / or the chassis of claim 4 or 5; Preferably, the method further comprises the step of introducing the expression cassette of claim 1 or 2 or the recombinant expression vector of claim 3 into the chassis of claim 4 or 5.

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