Genetic engineering strain, construction method thereof and application of genetic engineering strain in biosynthesis of 5-hydroxytryptophan

Abstract

The invention provides a genetic engineering strain, a construction method of the genetic engineering strain and application of the genetic engineering strain in biosynthesis of 5-hydroxytryptophan. According to the engineering bacterium, four key modules required by 5-HTP synthesis are sequentially introduced into a host genome in a genome integration mode through a CRISPR technology, and the four key modules comprise an aroGfbr-ppsA-tktA module for enhancing chorismate precursor supply, a trpEfbr-trpG module for catalyzing o-aminobenzoic acid generation, a salABCD gene cluster responsible for hydroxylating o-aminobenzoic acid into 5-hydroxy o-aminobenzoic acid, and a salABCD gene cluster responsible for hydroxylating 5-hydroxy o-aminobenzoic acid into 5-hydroxy o-aminobenzoic acid. And a trpDCBA module for converting the intermediate into the final product 5-HTP. The modules are respectively integrated into a plurality of non-essential gene regions, so that stable genetic expression is realized, and a complete metabolic pathway can be operated without plasmids or inducers. The strain can continuously accumulate 5-hydroxytryptophan (5-HTP) under a glucose fermentation condition, and has the advantages of simplicity and convenience in construction, high hereditary stability, low production cost, suitability for industrial-scale fermentation and the like.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering. Background Technology

[0002] 5-Hydroxytryptophan (5-HTP) is a direct precursor to the neurotransmitter serotonin and has significant applications in the treatment of depression, insomnia, and chronic headaches. Currently, 5-HTP is mainly extracted from Ghana seeds. This method is limited by plant sources, has a complex extraction process, is costly, and is easily affected by seasons and regions, making it difficult to meet market demand. Traditional chemical synthesis routes are lengthy, require stringent conditions, produce numerous byproducts, and have low yields, limiting their industrial-scale application. In recent years, the biosynthesis of 5-HAA using microbial metabolic engineering has become a mainstream trend.

[0003] Microbial synthesis offers a promising alternative for the sustainable production of 5-HTP. Previous studies have reported a pathway for synthesizing 5-HTP from glucose via 5-hydroxy-annaminobenzoic acid (5-HAA) using a "precursor-directed biosynthesis" strategy, expressed in *E. coli* with exogenous plasmids. The key to this pathway lies in utilizing salicylate 5-hydroxylase (S5H), which exhibits loose substrate selectivity, to convert annaminobenzoic acid (AA) to 5-HAA, followed by the conversion of 5-HAA to 5-HTP using the *E. coli* endogenous tryptophan synthase system (TrpDCBA).

[0004] However, plasmid-based expression systems have inherent drawbacks: plasmids are easily lost during passage, requiring the addition of antibiotics for maintenance, inducers increase costs, and the replication and expression of exogenous plasmids can impose a metabolic burden on host cells, affecting the genetic stability and production performance of the strain, which is not conducive to industrial-scale production. Summary of the Invention

[0005] The purpose of this invention is to overcome the instability and high cost of existing plasmid expression systems, and to provide a genetically stable, antibiotic- and inducer-free genetically engineered strain capable of efficiently synthesizing 5-HTP de novo, as well as its construction method.

[0006] This invention provides a genetically engineered strain for producing 5-hydroxytryptophan, which is constructed in an Escherichia coli host by stably inserting key genes of the 5-HTP biosynthetic pathway into specific locations on the chromosome through genome integration technology.

[0007] The 5-HTP biosynthetic pathway includes an upstream module and a downstream module:

[0008] Upstream module: Responsible for synthesizing the intermediate 5-hydroxy-an-aminobenzoic acid (5-HAA) from glucose. This module integrates the following genes:

[0009] Shikimate pathway enhancement genes: including at least the 3-deoxy-D-arabinohepenoic acid-7-phosphate synthase gene aroG that relieves feedback inhibition. fbr .

[0010] anthranilate synthase gene: encodes an enzyme with anthranilate synthase activity, preferably trpE from Escherichia coli. fbr G (feedback inhibition relief mutant).

[0011] Salicylic acid 5-hydroxylase gene: Encodes an enzyme capable of 5-hydroxylation using anthranilic acid (AA) as a non-natural substrate, preferably derived from the salABCD gene cluster of Ralstonia eutropha.

[0012] Downstream module: Responsible for converting 5-HAA into the final product 5-HTP. This module integrates:

[0013] Tryptophan biosynthesizing enzyme gene: Encodes a tryptophan synthase system that can utilize 5-HAA as a substrate, preferably the trpDCBA gene cluster from Escherichia coli.

[0014] To achieve stable and efficient expression of the pathway, this invention employs the following genome integration strategy:

[0015] Upstream module genes (e.g., trpE) fbr G and salABCD) integrate into pseudogene or non-essential gene sites in the host genome in multiple or single copy form. These sites include, but are not limited to: fhiA, rph, yeeL, yjiT, ygay, ilvG, or yciQ.

[0016] Integrating the downstream module gene (trpDCBA) into other independent pseudogene sites, such as rph and yeeL, can achieve physical separation and independent regulation from the upstream module, which helps reduce the loss of intermediate metabolites and improve the yield of 5-HTP.

[0017] Each integrated expression cassette is driven by a constitutive strong promoter (such as the lpp promoter, sequence shown in SEQ ID NO:1), ensuring sustained expression without the need for exogenous inducers.

[0018] In one specific implementation, the genetically engineered strain is obtained by sequentially integrating genes from the above pathways into designated chromosomal sites based on a strain of Escherichia coli capable of producing high levels of cladistic acid or anthranilic acid (e.g., the strain disclosed in CN114958890A) using genome editing technologies such as CRISPR-Cas9.

[0019] This invention also provides a method for constructing the genetically engineered strain, which mainly includes the following steps:

[0020] 1. Provide a host bacterium for Escherichia coli.

[0021] 2. Using the CRISPR-Cas9 system, upstream module gene expression cassettes (such as salABCD and trpE driven by the lpp promoter) are expressed. fbr G) Integrates into one or more sites in the host genome, such as fhiA, rph, yeeL, yjiT, ygay, ilvG, and yciQ, replacing the original pseudogene sequence.

[0022] 3. Using the CRISPR-Cas9 system, downstream module gene expression cassettes (such as trpDCBA driven by the lpp promoter) are integrated into the rph, yeeL and other sites of the host genome.

[0023] 4. Eliminate selection markers and editing tool plasmids to obtain genetically stable engineered strains free of exogenous plasmids.

[0024] This invention further provides the application of the genetically engineered strain in the production of 5-hydroxytryptophan. The application includes: inoculating the engineered strain into a fermentation medium with glucose as the primary or sole carbon source, and carrying out batch or fed-batch fermentation under aerobic conditions to produce and accumulate 5-HTP.

[0025] The beneficial effects of this invention are:

[0026] 1. High genetic stability: All pathway genes are integrated into the chromosome, eliminating the need for plasmids, thus eliminating the risk of plasmid loss. The strain has stable performance and is suitable for long-term subculturing and industrial fermentation.

[0027] 2. Low production cost: No antibiotics are needed to maintain plasmids, and no inducers are needed to induce gene expression, which simplifies the fermentation process and reduces raw material costs.

[0028] 3. Low metabolic burden: It avoids the huge metabolic pressure on the host cell caused by the multi-plasmid system, which is conducive to cell growth and efficient synthesis of target products.

[0029] 4. High production intensity: Through modular integration and promoter optimization, the synthesis pathway is coordinated and expressed in a coordinated manner, effectively guiding the carbon flow to 5-HTP synthesis, thereby improving yield and production intensity.

[0030] 5. Environmentally friendly: The entire production process does not require the use of chemical inducers and antibiotics, making it more green and sustainable. Attached Figure Description

[0031] Figure 1 This is a pathway diagram for the biosynthesis of 5-HTP provided by the present invention.

[0032] Figure 2 This is a shake-flask fermentation result of the integrated strain for producing 5-HTP provided in Example 2 of the present invention.

[0033] Figure 3 This is a graph showing the results of fed-batch fermentation of 5-HTP produced by the integrated strain provided in Example 3 of the present invention. Detailed Implementation

[0034] The present invention will be further described below with reference to the embodiments, but the present invention is not limited to the following embodiments.

[0035] Example 1: Construction of the genetically engineered strain SYT1

[0036] 1. Host strain: A modified Escherichia coli strain BW5 that can produce high levels of cleavage acid was selected as the starting strain for genome integration (see CN114958890A for its construction).

[0037] 2. Construction of integration vectors and fragments: Design and synthesize the salABCD gene cluster expression cassette driven by the lpp promoter (key part of the upstream module). Design and synthesize the trpDCBA gene cluster expression cassette driven by the lpp promoter (downstream module). For selected integration sites (e.g., fhiA for integrating salABCD; rph and yeeL for integrating trpDCBA), construct integration fragments containing approximately 500 bp homologous arms.

[0038] Construction of sgRNA expression plasmids targeting various integration sites (pTarget series): The target sequences used for sgRNA in this study are shown in Table 1:

[0039] Table 1. Target sequences used by sgRNA

[0040]

[0041] The primer sequences used in this study are shown in Table 2:

[0042] Table 2. Primer sequence listing

[0043]

[0044] P1 and P2 were used as a set of primers, and pTarget plasmid was used as a template. PCR was performed to obtain a nucleotide sequence containing sgRNA with a length of 2200 kbp. After agarose gel electrophoresis, the PCR product was purified and recovered using a gel recovery kit. The PCR purified solution was chemically transformed into E. coli DH5α competent cells, where it recombinated and self-ligated to form pTarget-fhiA plasmid with ampicillin resistance.

[0045] (3) Constructing the fhiA site integration fragment

[0046] The primer sequences used in this study are shown in Table 3.

[0047] Table 3. Primer sequence listing

[0048]

[0049] Using *E. coli* BW25113 as a template, primers were prepared in pairs (P3 and P4, P7 and P8) to amplify the two homologous arms of the gene *fhiA* using *E. coli* as a template. Using plasmid *pET-salABCD* as a template, primers were prepared in pairs (P5 and P6) to amplify the gene fragment *salABCD*. Then, using these three fragments as templates, primers were prepared in pairs (P3 and P8) to obtain an integrated fragment through PCR overlap extension. The integrated fragment was then subjected to agarose gel electrophoresis, and the PCR product was purified and recovered using a gel extraction kit.

[0050] (4) The pTarget-fhiA plasmid and the fhiA site integration fragment were introduced into the BW5-Cas9 strain in Example 2 (1) by electroporation. In the bacterial cells, sgRNA guided the Cas9 protein to recognize the integration site sequence and cut it. The bacterial cells' own repair function enabled the integration fragment to replace the pseudosense site through homologous recombination, resulting in a strain that replaced the fhiA gene on the genome with the gene salABCD promoted by the lpp promoter. The strain was then cultured at 30 degrees Celsius for 24 hours on a plate containing spectinomycin and ampicillin resistance.

[0051] (5) The above-mentioned genetically engineered strain was cultured in LB liquid medium containing spectinomycin, with 10 mmol / L arabinose added, and cultured at 30 degrees Celsius for 24 hours to induce Cas9 protein expression and degradation of the pTarget-fhiA plasmid. The resulting strain containing only the pCas plasmid was cultured in LB liquid medium containing spectinomycin.

[0052] (6) Eliminate pCas plasmid. Culture the genetically engineered bacteria that have had pTarget-fhiA plasmid eliminated in antibiotic-free liquid LB and culture at 42 degrees for 48 hours to lose the temperature-sensitive pCas plasmid. Verify the correct integration of each gene expression cassette by colony PCR and sequencing to obtain an engineered strain that does not carry any foreign plasmid.

[0053] (7) Obtain gene-integrated engineered strains that achieve stable inheritance without the need for antibiotics.

[0054] Constructing sgRNA plasmids for the rph and yeeL sites of the gene

[0055] The target sequences used for sgRNA in this study are shown in Table 4.

[0056] Table 4. Target sequences used by sgRNA at the rph and yeeL sites of genes

[0057]

[0058] The primer sequences used in this study are shown in Table 5.

[0059] Table 5 Primer sequence list

[0060]

[0061] Primers P9 and P2 were used as a pair, and primers P10 and P2 were used as a pair. Using pTarget plasmid as a template, PCR was performed to obtain nucleotide sequences containing sgRNA with a length of 2200 kbp. After agarose gel electrophoresis, the PCR products were purified and recovered using a gel extraction kit. The purified PCR solution was chemically transformed into E. coli DH5α competent cells, where it recombinated and self-ligated to form pTarget-rph and pTarget-yeeL plasmids containing ampicillin.

[0062] (7) Constructing the rph and yeeL site integration fragment

[0063] The primer sequences used in this study are shown in Table 6.

[0064] Table 6 Primer sequence list

[0065]

[0066]

[0067] Using *E. coli* BW25113 as a template, primers were prepared in pairs: P11 and P12, P15 and P16, P17 and P18, and P19 and P20. PCR amplification yielded two homologous arms each of the genes *rph* and *yeeL*. Using plasmid pCs-trpDCBA as a template, primers were prepared in pairs: P13 and P14. PCR amplification yielded the gene *trpDCBA* fragment. Then, using different pairs of homologous arms and gene fragments as templates, primers were prepared in pairs: P11 and P16, and P17 and P20. PCR overlap extension was used to obtain an integrated fragment. Agarose gel electrophoresis was performed, and the PCR products were purified and recovered using a gel extraction kit.

[0068] (8) The pTarget-rph plasmid and the rph gene site integration fragment were introduced into the strain in (5) of Example 2 after pTarget-fhiA was eliminated by electroporation. In the bacterial cells, sgRNA guided the cas9 protein to recognize the integration site sequence and cut it. The bacterial cells’ own repair function enabled the integration fragment to replace the pseudosense site by homologous recombination, resulting in a strain that replaced the rph gene on the genome with the trpDCBA gene. The strain was cultured at 30 degrees Celsius for 24 hours on a plate containing spectinomycin and ampicillin resistance.

[0069] (9) Remove the pTarget-rph plasmid to obtain bacteria containing only the pCas plasmid, using the same method as described in (5) of Example 2. This yields bacteria with the plasmid removed, containing only the pCas plasmid.

[0070] (10) The pTarget-yeeL plasmid and the yeeL gene site integration fragment were introduced into the strain in Example 2 (9) after pTarget-rph was eliminated by electroporation. In the bacterial cells, sgRNA guided the cas9 protein to recognize the integration site sequence and cut it. The bacterial cells' own repair function enabled the integration fragment to replace the pseudosense site by homologous recombination, resulting in a strain that replaced the yeeL gene on the genome with the gene trpDCBA promoted by the lpp promoter. The strain was cultured at 30 degrees Celsius for 24 hours on a plate containing spectinomycin and ampicillin resistance.

[0071] (11) Remove the pTarget-rph plasmid to obtain bacteria containing only pCas, as described previously. Obtain bacteria with the plasmid removed and containing only the pCas plasmid.

[0072] (12) Eliminate pCas plasmid. Culture the above-mentioned genetically engineered bacteria with pTarget-fhiA plasmid eliminated in antibiotic-free liquid LB. Culture at 42 degrees for 48 hours to lose the temperature-sensitive pCas plasmid. Verify the correct integration of each gene expression cassette by colony PCR and sequencing to obtain an engineered strain that does not carry any foreign plasmid.

[0073] Example 2: Production of 5-HTP by shake-flask fermentation

[0074] The integrated strain was streaked onto antibiotic-free LB agar plates and incubated overnight at 37°C. Single colonies were picked and inoculated into 5 mL LB tubes, and cultured at 37°C and 220 rpm for 10 h to prepare the seed culture. A 2% inoculum was then transferred to a 250 mL shake flask containing 50 mL of fermentation medium (composition: glucose 20 g / L, yeast extract 5 g / L, Na₂HPO₄ 6 g / L, KH₂PO₄ 3 g / L, NH₄Cl 1 g / L, NaCl 0.5 g / L, MgSO₄ 0.24 g / L, CaCl₂ 0.015 g / L, MOPS 2 g / L, pH 7.0). The culture was incubated with shaking at 30°C and 220 rpm for 72 h. Samples were taken every 12 h, the supernatant was collected by centrifugation, and the 5-HTP content was determined by HPLC.

[0075] Results: After 72 hours of fermentation, the yield of 5-HTP reached approximately 4.5 g / L.

[0076] Example 3: Feed-in batch fermentation for 5-HTP production

[0077] The seed culture of the integrated strain was inoculated into a 5L fermenter at a 5% inoculum. The initial fermentation medium was similar to that in shake flask media. The temperature was controlled at 30℃, pH 7.0 (adjusted with ammonia), and dissolved oxygen (DO) was maintained at approximately 20% by adjusting the stirring speed and aeration rate. When the initial glucose concentration (20 g / L) dropped below 10 g / L, a 600 g / L glucose solution was added, maintaining the residual glucose at approximately 10 g / L. The fermentation cycle was approximately 84 hours.

[0078] Results: Through fed-batch fermentation, the yield of 5-HTP reached approximately 25 g / L after 72 hours, with a carbon yield of 0.2 g / g, demonstrating good potential for industrial production.

[0079] in conclusion:

[0080] This invention successfully constructed a genetically stable 5-HTP-producing strain that requires neither inducers nor antibiotics through an innovative genome integration strategy. This strain efficiently converts glucose into 5-HTP, exhibiting excellent yields in both shake flask and fermenter environments. It provides a microbial manufacturing platform with significant application value for overcoming the bottlenecks of traditional 5-HTP production methods.

Claims

1. A genetically engineered strain that produces 5-hydroxytryptophan, characterized in that, The strain is *Escherichia coli*, and its genome contains a complete 5-hydroxytryptophan biosynthesis pathway through the integration of exogenous genes or overexpression of endogenous genes; the pathway includes at least: (i) An upstream synthesis module that catalyzes the production of glucose into the intermediate 5-hydroxy-o-aminobenzoic acid; (ii) A downstream synthesis module capable of catalyzing the formation of 5-hydroxy-o-aminobenzoic acid from 5-hydroxytryptophan; In this process, at least one or more sets of genes encoding key enzymes in the upstream and downstream synthesis modules are stably integrated into the chromosomal non-essential regions of the E. coli genome.

2. The genetically engineered strain according to claim 1, characterized in that, The upstream synthesis module includes a first enzyme with anthranilic acid synthase activity and a second enzyme with salicylate 5-hydroxylase activity; the downstream synthesis module includes a third enzyme with tryptophan biosynthesis activity, the third enzyme being capable of using 5-hydroxyanthranilic acid as a substrate.

3. The genetically engineered strain according to claim 2, characterized in that: The first enzyme having anthranilic acid synthase activity is selected from Escherichia coli. trpE and trpG At least one code in the gene is preferably insensitive to feedback inhibition. trpE fbr G Gene clusters; The second enzyme, possessing salicylate 5-hydroxylase activity, is selected from *Rawstoneella eutropha*. salABCD Gene cluster encoding; The third enzyme, which has tryptophan biosynthesis activity, is selected from Escherichia coli. trpDCBA Gene cluster encoding.

4. The genetically engineered strain according to any one of claims 1-3, characterized in that, The upstream synthesis module also includes a fourth enzyme assembly that enhances the throughput of the shikimic acid pathway, the fourth enzyme assembly including at least 3-deoxy-D-arabinohepenoic acid-7-phosphate synthase that relieves feedback inhibition.

5. The genetically engineered strain according to claim 1, characterized in that, The non-essential regions of the chromosome are selected from pseudogene loci or metabolically non-essential gene loci.

6. The genetically engineered strain according to claim 1, characterized in that, The genes encoding the key enzymes of all the synthetic modules were integrated into a gene selected from... fhiA , yjiT , ygay , ilvG , yciQ , rph , yeeL One or more sites in the middle.

7. The genetically engineered strain according to claim 6, characterized in that, trpE fbr G and salABCD Gene clusters are integrated into fhiA Site; the trpDCBA Gene clusters are integrated into rph and yeeL Site.

8. The genetically engineered strain according to claim 1, characterized in that, The strain was obtained by integrating the genes described in claims 1-7 using a host chassis of an Escherichia coli capable of producing cladistic acid or anthranilic acid through genome editing technology.

9. The genetically engineered strain according to any one of claims 1-8, characterized in that, Foreign genes or expression cassettes integrated into the genome are driven by constitutive strong promoters selected from... lpp Promoter.

10. A method for producing 5-hydroxytryptophan, characterized in that, The genetically engineered strain according to any one of claims 1-9 is cultured in a medium containing glucose as a carbon source to produce and accumulate 5-hydroxytryptophan.

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