A rhodioloside production strain, a construction method and application thereof

Abstract

The application provides a rhodioloside production strain and a construction method and application thereof, the strain lacks tyrR, feaB, ldhA, ushA, pykF genes, up-regulates tktA , aroG fbr 、 tyrA fbr、 pgm, galU, gntK the transcription level of a gene, and heterologously introduces a TDC gene from Lactobacillus brevis, a TYO gene from Micrococcus luteus, a ARO10 gene from Saccharomyces cerevisiae, and a ADH6 gene, heterologously introduces a GDH gene from Bacillus and a sugar transferase Oft8gt1 gene from Osmanthus fragrans; the construction method is to reconstruct the metabolic network of Escherichia coli to rhodioloside through metabolic engineering and genetic engineering means, which can effectively utilize glucose to synthesize rhodioloside from scratch, improve the production level of rhodioloside, and lay a solid foundation for the industrialized scale production thereof.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and fermentation engineering technology, and in particular to a rhodioloside-producing strain, its construction method, and its application. Background Technology

[0002] Rhodioloside ( salidroside Rhodiola rosea glycosides are natural phenylethanoid compounds with significant antioxidant, anti-fatigue, neuroprotective, and anti-hypoxia activities, and are widely used in the pharmaceutical, health, and cosmetic fields. In recent years, the efficient biosynthesis of rhodiola rosea glycosides by constructing microbial cell factories using synthetic biology strategies has become a research hotspot. Among them, *Escherichia coli* (E. coli) Escherichia coli Due to its clear genetic background, rapid growth, and ease of engineering modification, it is widely used as a chassis host for heterologous synthesis of rhodioloside.

[0003] However, current E. coli-based rhodioloside synthesis pathways generally rely on aromatic amino acid metabolism, typically requiring the derivation of erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP) from central carbon metabolism via the pentose phosphate pathway and glycolysis. These then proceed through multiple enzymatic reactions to generate the key precursor 4-hydroxyphenylpyruvate (4-HPP), which is subsequently decarboxylated by phenylpyruvate decarboxylase. ARO10, alcohol dehydrogenase ADH6 and Glycosyltransferase Oft8gt1 The catalytic production of the target product rhodioloside is a lengthy process (typically involving 6–9 or more exogenous or endogenous enzymes) with complex metabolic flux regulation.

[0004] More importantly, L-tyrosine, as a byproduct, accumulates readily intracellularly. On the one hand, key enzymes... tyrA The gene is subject to tyrosine feedback repression, limiting the supply of the precursor 4-HPP; on the other hand, through the... tyrA Site-directed mutagenesis of amino acids relieves tyrosine inhibition, but phenylpyruvate decarboxylase... ARO10The heterologous introduction of rhodioloside leads to restricted expression. 4-HPP readily converts to tyrosine under the action of endogenous transaminases, causing not only carbon loss and energy waste but also a metabolic burden on the bacteria, affecting strain activity. Simultaneously, a large amount of carbon is "locked" in intermediate metabolites such as tyrosine, unable to effectively flow to the final product, resulting in significant carbon loss and energy waste, severely restricting the yield and productivity of rhodioloside. Furthermore, the synthesis of aromatic compounds and the key precursor tyrosol, tyrosol, requires a large amount of NADPH. The host's endogenous NADPH regeneration capacity is limited, and blindly strengthening related metabolic pathways can lead to growth and metabolic disorders. Therefore, effectively addressing the balance between NADPH supply and metabolism while ensuring normal cellular physiology is crucial for achieving efficient microbial synthesis of rhodioloside. Thus, there is an urgent need to develop a novel metabolic engineering strategy that can shorten the synthetic pathway, reduce intermediate product accumulation, and increase NADPH supply to achieve efficient, stable, and high-yield synthesis of rhodioloside in *E. coli*. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a rhodioloside-producing strain.

[0006] Another technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned rhodioloside-producing strain.

[0007] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned rhodioloside-producing strain.

[0008] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: A rhodioloside-producing strain, named strain SRL15, is produced using Escherichia coli. E.coli W3110 was obtained as a chassis strain using a targeted modification method: [missing information] tyrR, feaB, ldhA, ushA, pykF Genes, upregulated tktA , aroG fbr , tyrA fbr , pgm, galU, gntK At the gene transcription level, heterologous introduction of Lactobacillus brevis ( Lactobacillus brevis (Source) TDC Genes, Micrococcus luteus ( Micrococcus luteus (Source) TYO Genes, brewer's yeast ( Saccharomyces cerevisiae (Source) ARO10 and ADH6 Genes, heterologously introduced into Bacillus ( Bacillus (Source) GDH Genes and Osmanthus ( Osmanthus fragrans ) Glycosyltransferases from Oft8gt1 Gene.

[0009] Preferably, the above-mentioned rhodioloside-producing strain has the following characteristics: [The following text appears to be incomplete and requires further context: "knockout of the strain genome"] tyrR, feaB, ldhA, ushA, pykF Genes; Enhancement using TRC promoters tktA Gene transcription and integration into the pykF gene locus in the genome; enhanced using the trc promoter. aroG fbr Gene transcription was performed and integrated into the feaB and yeeL pseudogene sites in the genome, respectively; the trc promoter was used for reinforcement. tyrA fbr Gene transcription and integration into the genomic yeeP pseudogene site; enhancement of Lactobacillus brevis-derived genes using the lac promoter. TDC Gene transcription and integration into the genomic ycdN pseudogene site; enhancement of Micrococcus luteus-derived genes using the lac promoter. TYO Genes were integrated into the yjgX pseudogene locus in the genome; the trc promoter was used to enhance the *Saccharomyces cerevisiae*-derived gene. ARO10 Genes were integrated into the fhiA pseudogene locus in the genome; the trc promoter was used to enhance the *Saccharomyces cerevisiae*-derived gene. ADH6 Genes were integrated into the yjiP pseudogene locus in the genome; the lac promoter was used to enhance Bacillus-derived genes. GDH Genes were extracted and integrated into the yncI pseudogene locus in the genome; enhancement was achieved using the trc promoter. pgm Genes were extracted and integrated into the molR pseudogene site in the genome; enhancement was achieved using the trc promoter. galU Genes were extracted and integrated into the ylbE pseudogene locus in the genome; enhancement was achieved using the trc promoter. gntK The gene was integrated into the mbhA pseudogene locus in the genome; the trc promoter was used to enhance the osmanthus-derived gene. Oft8gt1 The genes were integrated into the ushA and ygaY pseudogene loci of the genome, respectively.

[0010] Preferably, the above-mentioned rhodioloside-producing strain, wherein aroG fbr The gene underwent a site-directed mutation, where all bases at position 436 were changed from G to A, resulting in the amino acid residue at position 146 changing from aspartic acid (D) to asparagine (N). tyrA fbr The gene underwent site-directed mutation, changing G to A at position 159 and C to T at position 1061, that is, mutating methionine (M) to isoleucine (I) and alanine (A) to valine (V).

[0011] Preferably, the above-mentioned rhodioloside-producing strain is *Escherichia coli*, the originating strain. E.coli W3110 is E.coli W3110ATCC 27325 (commercially available strain).

[0012] Preferably, the above-mentioned rhodioloside-producing strain, wherein tyrR The nucleotide sequence of the gene is shown in SEQ ID NO.1. feaB The nucleotide sequence of the gene is shown in SEQ ID NO.2 of the sequence listing. ldhA The nucleotide sequence of the gene is shown in SEQ ID NO.3 of the sequence listing. ushA The nucleotide sequence of the gene is shown in SEQ ID NO.4 of the sequence listing. pykF The nucleotide sequence of the gene is shown in SEQ ID NO.5 of the sequence listing. tktA The nucleotide sequence of the gene is shown in SEQ ID NO.6 of the sequence listing. aroG fbr The nucleotide sequence of the gene is shown in SEQ ID NO.7 of the sequence listing. tyrA fbr The nucleotide sequence of the gene is shown in SEQ ID NO.8 of the sequence listing. pgm The nucleotide sequence of the gene is shown in SEQ ID NO.9 of the sequence listing. galU The nucleotide sequence of the gene is shown in SEQ ID NO.10 of the sequence listing. gntK The nucleotide sequence of the gene is shown in SEQ ID NO.11 of the sequence listing. TDC The nucleotide sequence of the gene is shown in SEQ ID NO.12 of the sequence listing. TYO The nucleotide sequence of the gene is shown in SEQ ID NO. 13 of the sequence listing. ARO10 The nucleotide sequence of the gene is shown in SEQ ID NO.14 of the sequence listing. ADH6 The nucleotide sequence of the gene is shown in SEQ ID NO.15 of the sequence listing. GDH The nucleotide sequence of the gene is shown in SEQ ID NO.16 of the sequence listing. Oft8GT1 The nucleotide sequence of the gene is shown in SEQ ID NO.17, the nucleotide sequence of the trc promoter is shown in SEQ ID NO.18, and the nucleotide sequence of the lac promoter is shown in SEQ ID NO.19.

[0013] The above-mentioned method for constructing rhodioloside-producing strains involves starting with the strain... E.coli The following are the specific steps for targeted modification based on the W3110: (1) Knockout of repressor regulatory protein genes on the genome tyrR ; (2) Enhancement of Saccharomyces cerevisiae-derived yeast using the TRC promoter ARO10 Gene; (3) Enhancement of Saccharomyces cerevisiae-derived yeast using the TRC promoter ADH6 Gene; (4) Enhance the osmanthus-derived osmanthus source using the trc promoter. Oft8gt1 Genes, on Oft8gt1 The gene is copied twice; (5) Knockout of the gene encoding UDP-glucose hydrolase in the genome ushA Genes and pyruvate phosphotransferase pykF Gene; (6) Enhancement using the trc promoter tktA Gene transcription; (7) The gene encoding hydroxycarboxylic acid dehydrogenase was knocked out in the genome. ldhA Genes and encoding phenylacetaldehyde dehydrogenase feaB Gene; (8) Enhancement using the trc promoter aroG fbr Gene transcription, on aroG fbr The gene is copied twice; (9) Strengthening using the trc promoter tyrA fbr Gene transcription; (10) Utilizing the lac promoter to enhance the efficacy of Lactobacillus brevis-derived probiotics TDC Gene transcription; (11) Using the lac promoter to enhance the efficacy of Micrococcus luteus-derived probiotics TYO Gene; (12) Enhancement using the trc promoter gntK Gene; (13) Using the lac promoter to enhance Bacillus-derived [products / products] GDH Gene; (14) Enhancement using the trc promoter pgm Gene; (15) Enhancement using the trc promoter galU Gene.

[0014] Preferably, in the method for constructing the above-mentioned rhodioloside-producing strain, the starting strain... E.coli The following are the specific steps for targeted modification based on the W3110: (1) Knockout of repressor regulatory protein genes on the genome tyrR ; (2) Introducing an enzyme encoding phenylpyruvate decarboxylase from Saccharomyces cerevisiae at the fhiA pseudogene locus in the genome. ARO10 Genes, and are initiated by the trc promoter; (3) Introducing an alcohol dehydrogenase derived from Saccharomyces cerevisiae into the yjiP pseudogene locus in the genome. ADH6Genes, and are initiated by the trc promoter; (4) Introducing a gene encoding glycosyltransferase at the ygaY pseudogene locus in the genome. Oft8gt1 Genes, and are initiated by the trc promoter; (5) The enzyme encoding UDP-glucose hydrolase was knocked out in the genome. ushA The gene integrates a glycosyltransferase at this point. Oft8gt1 Gene; (6) Knockout of pyruvate phosphotransferase in the genome pykF The gene, and at this site, the transketolase gene is integrated. tktA Boot using the trc bootloader; (7) The gene encoding hydroxycarboxylic acid dehydrogenase was knocked out in the genome. ldhA Gene; (8) The gene encoding phenylacetaldehyde dehydrogenase was knocked out in the genome. feaB The gene was inserted at that point to relieve feedback inhibition of 3-deoxy-7-phosphate heptanuronate synthase. aroG fbr The gene was started using the Trc promoter; a 3-deoxy-7-phosphate heptanuronate synthase that relieves feedback inhibition was introduced at the yeeL pseudogene site in the genome. aroG fbr Genes are started using the Trc promoter; (9) Introducing a gene encoding prebenzoic acid dehydrogenase at the yeeP pseudogene locus in the genome. tyrA fbr Genes, and are initiated by the trc promoter; (10) Introducing a tyrosine decarboxylase derived from Lactobacillus brevis into the ycdN pseudogene locus in the genome. TDC Genes, and are promoted by the promoter lac; (11) Introducing a gene encoding tyrosine oxidase derived from Micrococcus luteus into the yjgX pseudogene locus of the genome. TYO Genes, and are promoted by the promoter lac; (12) Introducing gluconate kinase into the mbhA pseudogene locus in the genome gntK Genes are started using the trc promoter; (13) Introduce glucose dehydrogenase from Bacillus at the yncI pseudogene locus in the genome. GDH Genes, and are initiated by the lac promoter; (14) Introducing a gene encoding phosphoglucomutase into the molR pseudogene locus in the genome. pgm Genes, and are initiated by the trc promoter; (15) Introducing a gene encoding UDP-glucose pyrophosphorylase at the ylbE pseudogene site in the genome. galU Genes, and are initiated by the trc promoter.

[0015] Application of the above-mentioned rhodioloside-producing strains in the production of rhodioloside.

[0016] Preferably, the above-mentioned application involves producing rhodioloside using a fermentation method in a shake flask or fermenter.

[0017] Preferably, the above application follows these steps: (1) Slant culture: The rhodioloside-producing strain was inoculated onto a slant culture medium and cultured at 36-37℃ for 12-16h; (2) Shake flask seed culture: Take solid slant inoculum and inoculate it into shake flask culture medium for fermentation. The culture temperature is 36-37℃, the culture time is 12-20h, the shaking speed is 200-240r / min, and the pH is 6.4-6.7. (3) Shake flask fermentation culture: the fermentation inoculum amount is 1-5%, the culture temperature is 32-34℃, the pH is 6.4-6.7, the culture time is 24-36h, and the shaking speed is 180-220r / min.

[0018] Preferably, in the above application, the slant culture medium used in step (1) is the general LB solid culture medium.

[0019] Preferably, in the above application, the seed culture medium used in step (2) is: glucose 20-30g / L, glycerol 20-30g / L, yeast extract powder 3-5g / L, peptone 1.2-1.5g / L, anhydrous magnesium sulfate 1-2g / L, potassium dihydrogen phosphate 1-3g / L, ammonium sulfate 1-2g / L, ferrous sulfate heptahydrate 20-40mg / L, manganese sulfate monohydrate 10-20mg / L, and the remainder is water.

[0020] Preferably, in the above application, the fermentation culture medium used in step (3) is: yeast extract powder 4-5 g / L, peptone 2-3 g / L, corn steep liquor 3 g / L, anhydrous magnesium sulfate 1.5-2 g / L, potassium dihydrogen phosphate 1-3 g / L, ammonium sulfate 1-2 g / L, ferrous sulfate heptahydrate 20-40 mg / L, manganese sulfate monohydrate 10-20 mg / L, VB1, VB3, and VB5 each 2-3 mg / L, and the remainder is water.

[0021] All of the above-mentioned culture media can be prepared using standard methods.

[0022] Beneficial effects: The aforementioned rhodioloside-producing strain introduces a dual synthetic pathway for rhodioloside. By optimizing the reducing power supply system, it effectively improves the synthesis efficiency of rhodioloside, exhibiting excellent synthetic capacity and stable performance. It utilizes inexpensive carbon sources for de novo synthesis, without nutritional deficiencies, plasmids, or byproducts. Its construction method involves reconstructing the metabolic network from *E. coli* to rhodioloside through metabolic and genetic engineering, enabling efficient de novo synthesis of rhodioloside from glucose, thus improving the production level of rhodioloside and laying a solid foundation for its industrial-scale production. Specifically: (1) In the starting strain E.coli Knockout in the W3110 genome feaB Genes and ushA The gene blocked the degradation pathway of rhodioloside, which reduced carbon loss and promoted the accumulation of rhodioloside while maintaining normal bacterial growth.

[0023] (2) By knocking out tyrR Genes that enhance the expression activity of key enzymes; by knocking out pykF , expressed tktA The gene is enriched with precursors for the synthesis of aromatic products; by knocking out ldhA Genes that prevent pyruvate from flowing to lactate ensure normal bacterial growth and metabolism; overexpression of aroG fbr Genes that significantly increase the metabolic flux of the shikimic acid pathway; overexpression tyrA fbr The gene eliminates the feedback inhibition caused by tyrosine, allowing more metabolic flux to be directed to the rhodioloside precursor 4-HPP. Due to the difficulty in rhodioloside synthesis, this application innovatively constructs a dual synthetic pathway for rhodioloside to enhance its accumulation. Specifically, although tyrosine competes for 4-HPP, to avoid the nutritional deficiency of bacterial tyrosine caused by knockout, a heterologous gene from *Lactobacillus brevis* is introduced. TDC Genes and Micrococcus luteus origin TYO The gene was modified and its expression was optimized through promoter engineering to redirect the carbon flow to tyrosine to 4-HPAAL, thus constructing a pathway for the synthesis of rhodioloside from tyrosine; heterologous introduction of yeast-derived rhodioloside... ARO10 and ADH6 Genes, the origin of osmanthus Oft8gt1 Genes were used to construct a pathway for the synthesis of rhodioloside from 4-hydroxyphenylpyruvic acid. This dual-synthetic pathway directs all metabolic flux into the rhodioloside synthesis pathway, avoiding carbon loss and eliminating nutrient deficiencies in the bacterium, thus effectively improving the synthesis efficiency of rhodioloside.

[0024] (3) The cofactor supply system of the engineered bacteria was optimized by overexpressing gluconate kinase. gntK Genes, heterologous introduction of Bacillus-derived genes GDHGenes that increase NAD(P)H supply, thus supporting NAD(P)H-dependent genes. ADH6 Gene expression provided sufficient cofactors. The resulting rhodioloside engineered strain SRL15 used glucose as a carbon source, and the fermentation process required no addition of rhodioloside precursors or antibiotics, demonstrating promising prospects for industrial application. Attached Figure Description

[0025] Figure 1 This is a diagram illustrating the construction method of the rhodioloside-producing strain SRL15 described in this invention. Detailed Implementation

[0026] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0027] Unless otherwise specified, the percentage sign "%" in the examples refers to the mass percentage. The percentage of a solution refers to the number of grams of solute contained in 100 mL. The percentage between liquids refers to the volume ratio of the solution at 25°C.

[0028] The starting strain used in the examples was wild-type. E.coli W3110 ATCC 27325 (commercially available), the corresponding promoter and gene are shown in the sequence listing. Primers used in the construction of the involved strains are shown in Table 1.

[0029] Table 1 Primers used in strain construction

[0030]

[0031]

[0032]

[0033]

[0034]

[0035]

[0036]

[0037] The gene editing methods used in the examples refer to the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genomeediting. Metabolic Engineering, 2015, 31: 13-21.). Unless otherwise specified, all technical terms used in this invention are explained in this article. In this invention, "knockout" refers to the inactivation of the target gene, and "introduction" refers to the insertion of a foreign gene into the engineered bacterial genome after linking it with a promoter and terminator.

[0038] Example 1 This embodiment aims to illustrate the specific construction steps of strain SRL15. In particular, if there are similar gene manipulation methods in the embodiment, they will only be provided once and annotated, without further elaboration.

[0039] (1) Knockout tyrR Genes: E.coli Using the W3110 genome as a template, upstream and downstream homologous arms were amplified by PCR using tyrR-US, tyrR-UA, tyrR-DS, and tyrR-DA, respectively. Then, using these homologous arms as templates, overlapping fragments were amplified by PCR using tyrR-US and tyrR-DA primers. Annealing was then performed using pGRB-tyrR-S and pGRB-tyrR-A primers to obtain gRNA fragments, which were then ligated into the pGRB vector to obtain tyrR-pGRB. Preparation... E.coli W3110 electrotransformed competent cells, and the overlapping fragment was electrotransformed together with tyrR-pGRB into competent cells. Positive transformants were then screened to obtain strain SRL1.

[0040] (2) Control at the fhiA pseudogene site using the trc promoter ARO10 Gene overexpression: (E.) coli Using the W3110 genome as a template, upstream and downstream homologous arms and gRNA fragments were obtained by PCR amplification of fhiA-US, fhiA-UA, fhiA-DS, fhiA-DA, pGRB-fhiA-S, and pGRB-fhiA-A, respectively. Then, [the following was used]... S.cerevisiaeUsing the SCY4741 genome as a template, the target gene fragment was obtained using primers fhiA-ARO10-S and fhiA-ARO10-A. Using upstream and downstream homologous arms and the target fragment Ptrc-ARO10 as templates, and with primers fhiA-US and fhiA-DA as primers, overlapping fragments were amplified by overlapping PCR. Using primers pGRB-fhiA-S and pGRB-fhiA-A as primers, the gRNA fragment was annealed and ligated into the pGRB vector to obtain fhiA-pGRB. E. coli W3110 electroporated competent cells, and the overlapping fragment was electroporated together with fhiA-pGRB into competent cells. Positive transformants were obtained by screening, and the resulting strain was named SRL2.

[0041] (3) Use the trc promoter to control the yjiP pseudogene site. ADH6 Gene overexpression: This involves the same procedure as in step (2), except that it uses... S.cerevisiae The target gene fragment was obtained using the SCY4741 genome as a template, with primers yjiP-ADH6-S and yjiP-ADH6-A used. E.coli The upstream homologous arm, downstream homologous arm, and gRNA fragment of the W3110 genome were obtained using primers yjiP-US, yjiP-UA, yjiP-DS, yjiP-DA, pGRB-yjiP-S, and pGRB-yjiP-A, resulting in strain SRL3.

[0042] (4) Use the trc promoter to control the ygaY pseudogene site. Oft8gt1 Gene overexpression: This involves the same procedure as step (2), except that the target gene fragment is obtained through artificial synthesis, using primers ygaY-Oft8gt1-S and ygaY-Oft8gt1-A; E.coli The upstream homologous arm, downstream homologous arm, and gRNA fragment of the W3110 genome were obtained using primers ygaY-US, ygaY-UA, ygaY-DS, ygaY-DA, pGRB-ygaY-S, and pGRB-ygaY-A, resulting in strain SRL4.

[0043] (5) Knockout ushA Genes are integrated at this site. Oft8gt1 Gene: The procedure is the same as in step (1), except that the primers used are ushA-US, ushA-UA, ushA-DS, ushA-DA, pGRB-ushA-S, and pGRB-ushA-A. Next, using upstream and downstream homologous arms as templates, and ushA-US and ushA-DA as primers, and artificially synthesized... Oft8gt1The gene fragment was amplified by overlapping PCR to obtain an overlapping fragment; using pGRB-ushA-S and pGRB-ushA-A as primers, the gRNA fragment was annealed and ligated with the pGRB vector to obtain ushA-pGRB; competent cells were prepared, and the overlapping fragment and uhsA-pGRB were electroporated into competent cells, and positive transformants were screened to obtain competent cells and strain SRL5 was obtained.

[0044] (6) Knockout pykF Gene: The procedure is the same as in step (1), except that the primers used are pykF-US, pykF-UA, pykF-DS, pykF-DA, pGRB-pykF-S, and pGRB-pykF-A. Using upstream and downstream homologous arms as templates, and pykF-US and pykF-DA as primers, and with... E.coli Using the W3110 genome as a template and tktA-S and tktA-A as primers, the resulting amplification was achieved via PCR. tktA The gene fragment was amplified by overlapping PCR to obtain the pykF-tktA overlapping fragment; using pGRB-pykF-S and pGRB-pykF-A as primers, the gRNA fragment was annealed and ligated with the pGRB vector to obtain pykF-pGRB; competent cells were prepared, and the overlapping fragment and pykF-pGRB were electroporated into competent cells, and positive transformants were screened to obtain competent cells and obtain strain SRL6.

[0045] (7) Knockout ldhA Gene: It has the same operation method as in step (1), the difference being that the primers used are l dhA-US, ldhA-UA, ldhA-DS, ldhA-DA, pGRB-ldhA-S, pGRB-ldhA-A, and strain SRL7 was obtained.

[0046] (8) Knockout feaB Gene: The same operation method as in step (1) is used, except that the primers used are feaB-US, feaB-UA, feaB-DS, feaB-DA, pGRB-feaB-S, and pGRB-feaB-A. Upstream and downstream homologous arms are obtained by PCR amplification. Then, the mutant nucleotide sequence is designed on aroGtb-A and aroGtb-S, and there are homologous sequences. PCR amplification is performed using primers aroG-S / aroGtb-A and aroGtb-S / aroG-A to obtain fragment 1 and fragment 2, respectively. Then, using fragment 1 and fragment 2 as templates, overlap PCR is performed using primers aroG-S / aroG-A to obtain the target fragment Ptrc-aroG. fbr; using upstream and downstream homologous arms and the target fragment Ptrc-aroG fbr Using feaB-US and feaB-DA as primers, overlapping fragments were amplified by overlapping PCR; gRNA fragments were obtained by annealing using pGRB-feaB-S and pGRB-feaB-A primers, and then ligated into the pGRB vector to obtain feaB-pGRB; E was prepared. coli W3110 electroporated competent cells, and the overlapping fragment was electroporated together with feaB-pGRB into competent cells. Positive transformants were selected, and competent cells were created. The trc promoter was used to control the yeeL pseudogene site. aroG fbr Gene overexpression: (E.) coli Using the W3110 genome as a template, upstream and downstream homologous arms were obtained by PCR amplification using yeeL-US, yeeL-UA, yeeL-DS, and yeeL-DA, respectively. Then, the mutant nucleotide sequences were designed onto aroGtb-A and aroGtb-S, with homologous sequences. Fragment 1 and fragment 2 were obtained by PCR amplification using primers aroG-S / aroGtb-A and aroGtb-S / aroG-A, respectively. Fragment 1 and fragment 2 were then used as templates and amplified using primer yeeL-aroG... fbr -S / yeeL-aroG fbr -A was used for overlap PCR to obtain the target fragment Ptrc-aroG. fbr ; using upstream and downstream homologous arms and the target fragment Ptrc-aroG fbr Using yeeL-US and yeeL-DA as templates, overlapping fragments were amplified by overlapping PCR; using pGRB-yeeL-S and pGRB-yeeL-A as primers, gRNA fragments were annealed to obtain gRNA fragments, which were then ligated into the pGRB vector to obtain yeeL-pGRB; E was prepared. coli W3110 electrotransformed competent cells, and the overlapping fragment was electrotransformed together with yeeL-pGRB into competent cells. Positive transformants were obtained by screening, and the resulting strain was named strain SRL8.

[0047] (9) Use the trc promoter to control the yeeP pseudogene site. tyrA fbr Gene overexpression: tyrA is controlled at the yeeP gene locus using the trc promoter. fbr Gene overexpression: (E.) coliUsing the W3110 genome as a template, upstream and downstream homologous arms were obtained by PCR amplification using yeeP-US, yeeP-UA, yeeP-DS, and yeeP-DA, respectively. Then, the mutant nucleotide sequences were designed onto tyrAtb-1A and tyrAtb-2S, with homologous sequences. Fragment 1 and fragment 2 were obtained by PCR amplification using primers tyrA-S / tyrAtb-1A and tyrAtb-2S / tyrAtb-3A, respectively. Finally, using fragment 1 and fragment 2 as templates, primer yeeP-tyrA... fbr -S / yeeP-tyrA fbr -A was used for overlap PCR to obtain the target fragment Ptrc-tyrA fbr ; using upstream and downstream homologous arms and the target fragment Ptrc-tyrA fbr Using yeeP-US and yeeP-DA as templates, overlapping fragments were amplified by overlapping PCR; gRNA fragments were obtained by annealing using pGRB-yeeP-S and pGRB-yeeP-A as primers, and then ligated into the pGRB vector to obtain yeeP-pGRB; E was prepared. coli W3110 was used to electrotransform competent cells, and the overlapping fragment was electrotransformed together with yeeP-pGRB into competent cells. Positive transformants were obtained by screening, and the resulting strain was named SRL9.

[0048] (10) Control at the ycdN pseudogene site using the lac promoter TDC Gene overexpression: This involves the same procedure as step (2), except that the target gene fragment is obtained using the artificially synthesized gene TDC as a template, and the primers used are ycdN-TDC-S and ycdN-TDC-A; E.coli The upstream homologous arm, downstream homologous arm, and gRNA fragment were obtained from the W3110 genome using primers ycdN-US, ycdN-UA, ycdN-DS, ycdN-DA, pGRB-ycdN-S, and pGRB-ycdN-A. Strain SRL10 was then obtained.

[0049] (11) Control at the yjgX pseudogene locus using the lac promoter TYO Gene overexpression: This involves the same procedure as step (2), except that the target gene fragment is obtained using the artificially synthesized gene TYO as a template, and the primers used are yjgX-TYO-S and yjgX-TYO-A; E.coliThe upstream and downstream homologous arms and gRNA fragments of the W3110 genome were obtained using primers yjgX-US, yjgX-UA, yjgX-DS, yjgX-DA, pGRB-yjgX-S, and pGRB-yjgX-A. The competent cells were SRL8, and strain SRL11 was obtained.

[0050] (12) Control at the mbhA pseudogene locus using the lac promoter gntK Gene overexpression: This involves the same procedure as in step (2), except that E is used. coli The target gene fragment was obtained using the W3110 genome as a template, with primers mbhA-gntK-S and mbhA-gntK-A used; E. coli The upstream homologous arm, downstream homologous arm, and gRNA fragment of the W3110 genome were obtained using primers mbhA-US, mbhA-UA, mbhA-DS, mbhA-DA, pGRB-mbhA-S, and pGRB-mbhA-A, resulting in strain SRL12.

[0051] (13) Control at the yncI pseudogene site using the lac promoter GDH Gene overexpression: The procedure is the same as in step (2), except that the target gene fragment is obtained using the genome of Bacillus subtilis subsp. subtilis 168: BSU03930 as a template, and the primers used are yncI-GDH-S and yncI-GDH-A; E.coli The upstream homologous arm, downstream homologous arm, and gRNA fragment of the W3110 genome were obtained using primers yncI-US, yncI-UA, yncI-DS, yncI-DA, pGRB-yncI-S, and pGRB-yncI-A, resulting in strain SRL13.

[0052] (14) Control at the molR pseudogene site using the trc promoter pgm Gene overexpression: This involves the same procedure as in step (2), except that it uses... E.coli The target gene fragment was obtained using the W3110 genome as a template, with primers molR-pgm-S and molR-pgm-A used. E.coli The upstream homologous arm, downstream homologous arm, and gRNA fragment of the W3110 genome were obtained using primers molR-US, molR-UA, molR-DS, molR-DA, pGRB-molR-S, and pGRB-molR-A, resulting in strain SRL14.

[0053] (15) Use the trc promoter to control the ylbE pseudogene site.galU Gene overexpression: This involves the same procedure as in step (2), except that it uses... E.coli The target gene fragment was obtained using the W3110 genome as a template, with primers ylbE-galU-S and ylbE-galU-A used. E.coli The upstream homologous arm, downstream homologous arm, and gRNA fragment of the W3110 genome were obtained using primers ylbE-US, ylbE-UA, ylbE-DS, ylbE-DA, pGRB-ylbE-S, and pGRB-ylbE-A, resulting in strain SRL15.

[0054] Example 2 This embodiment aims to illustrate the application of the strain constructed in Example 1 in shake-flask fermentation. The specific steps are as follows: ① Slant culture: Take the bacterial strain preserved in the -80℃ refrigerator and inoculate it on the slant culture medium. Incubate at 36-37℃ for 14 h. The slant culture medium is the general LB solid medium (5g / L yeast extract, 10g / L peptone, 10g / L sodium chloride, 15g / L agar powder, and the rest is water).

[0055] ② Shake-flask seed culture: Inoculate solid slant culture into shake-flask culture medium for fermentation. The culture temperature is 36-37℃, the culture time is 16 hours, the shaking speed is 220 r / min, and the pH is 6.4-6.7. The seed culture medium used is: glucose 25 g / L, glycerol 25 g / L, yeast extract 4 g / L, peptone 1.3 g / L, anhydrous magnesium sulfate 1.5 g / L, potassium dihydrogen phosphate 2.5 g / L, ammonium sulfate 1.5 g / L, ferrous sulfate heptahydrate 30 mg / L, manganese sulfate monohydrate 15 mg / L, and the remainder is water.

[0056] ③ Shake-flask fermentation culture: The inoculum size is 5%, the culture temperature is 34℃, the pH is 6.7, the culture time is 30h, and the shaker speed is adjusted in stages, 0-20h: when OD 600 When the concentration reaches 0.5, add 0.1 mmol / L of IPTG inducer and rotate at 220 rpm. After 20-30 hours, rotate at 180 rpm. The fermentation medium used is: yeast extract 4.5 g / L, peptone 2.5 g / L, corn steep liquor 3 g / L, anhydrous magnesium sulfate 1.8 g / L, potassium dihydrogen phosphate 2.5 g / L, ammonium sulfate 1.5 g / L, ferrous sulfate heptahydrate 35 mg / L, manganese sulfate monohydrate 15 mg / L, VB1, VB3, and VB5 2.5 mg / L each, with the remainder being water.

[0057] Wild-type E. coli W3110 served as the control group. After 36 hours of fermentation, wild-type E was confirmed. coliW3110 failed to accumulate rhodioloside. The results of rhodioloside yield by the strain constructed in Example 1 during shake-flask fermentation are shown in Table 2. Among them, the engineered strain SRL15 accumulated 1.26 g / L of rhodioloside.

[0058] Table 2. Effects of key metabolic modification sites on rhodioloside yield during shake-flask fermentation

[0059] This invention utilizes systematic metabolic engineering to construct an engineered strain that produces rhodioloside. To verify the role of each key gene modification in this strategy, as shown in Table 2, the modified strain was constructed using a basic rhodioloside synthesis module (…). aro10 / adH6 and Oft8gt1 Based on the rhodioloside-producing strains, blocking the competitive pathway ( ΔpykF / ΔldhA / ΔfeaB / ΔushA, pgm and galU ), and release feedback inhibition ( aroG fbr / tyrA fbr ), constructing plasmids and introducing TDC / TYO pathways and enhance NADPH supply ( GDH / gntK The yield of rhodioloside in shake flasks was increased from 0.16 g / L to 1.26 g / L, which is about 7.9 times higher than that of the baseline glycoside-producing strain. The yield results indicate that the above metabolic engineering modification can synergistically promote precursor supply, reducing power supply and glycosylation efficiency, thereby increasing the yield of rhodioloside.

[0060] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention. Improvements and modifications such as strain modification based on the method of the present invention or based on the method are all considered to be within the scope of protection of the present invention.

Claims

1. A rhodioloside-producing strain, characterized in that: E. coli E. coli W3110 was obtained as a chassis strain using a targeted modification method: [missing information] tyrR, feaB, ldhA, ushA, pykF Genes, upregulated tktA , aroG fbr 、 tyrA fbr pgm, galU, gntK At the transcriptional level, heterologous introduction of Lactobacillus brevis-derived genes... TDC Genes, Micrococcus luteus-derived TYO Genes, brewer's yeast ARO10 and ADH6 Genes were heterologously introduced from Bacillus. GDH Genes and osmanthus-derived glycosyltransferases Oft8gt1 Gene, wherein, the TDC The nucleotide sequence of the gene is shown in SEQ ID NO. 12 of the sequence listing. TYO The nucleotide sequence of the gene is shown in SEQ ID NO.13 of the sequence listing. ARO10 The nucleotide sequence of the gene is shown in SEQ ID NO.14 of the sequence listing. ADH6 The nucleotide sequence of the gene is shown in SEQ ID NO.15 of the sequence listing. GDH The nucleotide sequence of the gene is shown in SEQ ID NO.16 of the sequence listing. Oft8GT1 The nucleotide sequence of the gene is shown in SEQ ID NO. 17 of the sequence listing.

2. The rhodioloside-producing strain according to claim 1, characterized in that: Knockout of the strain genome tyrR, feaB, ldhA, ushA, pykF Gene; Strengthening using TRC promoters tktA Gene transcription and integration into the pykF gene locus in the genome; enhanced using the trc promoter. aroG fbr Gene transcription, and integration into the feaB and yeeL pseudogene loci in the genome, respectively; Strengthening using TRC promoters tyrA fbr Gene transcription and integration into the yeeP pseudogene locus in the genome; Enhanced Lactobacillus brevis-derived probiotics using the lac promoter TDC Gene transcription and integration into the ycdN pseudogene locus in the genome; Enhancement of Micrococcus luteus-derived strains using the lac promoter TYO Genes are integrated into the yjgX pseudogene locus in the genome; Enhancement of Saccharomyces cerevisiae source using TRC promoter ARO10 Genes are integrated into the fhiA pseudogene locus in the genome; Enhancement of Saccharomyces cerevisiae source using TRC promoter ADH6 Genes are integrated into the genome at pseudogene loci; Enhancement of Bacillus-derived [products] using the lac promoter GDH Genes are integrated into the yncI pseudogene locus in the genome; Strengthening using TRC promoters pgm Genes are integrated into the molR pseudogene locus in the genome; Strengthening using TRC promoters galU Genes are integrated into the ylbE pseudogene locus in the genome; Strengthening using TRC promoters gntK Genes are integrated into the mbhA pseudogene locus in the genome; Enhance the source of Osmanthus using TRC promoter Oft8gt1 The genes were integrated into the ushA and ygaY pseudogene loci of the genome, respectively.

3. The rhodioloside-producing strain according to claim 1, characterized in that: The E. coli E. coli W3110 is E. coli W3110 ATCC 27325.

4. The rhodioloside-producing strain according to claim 1 or 2, characterized in that: The tyrR The nucleotide sequence of the gene is shown in SEQ ID NO.1 of the sequence listing. feaB The nucleotide sequence of the gene is shown in SEQ ID NO.2 of the sequence listing. ldhA The nucleotide sequence of the gene is shown in SEQ ID NO.3 of the sequence listing. ushA The nucleotide sequence of the gene is shown in SEQ ID NO.4 of the sequence listing. pykF The nucleotide sequence of the gene is shown in SEQ ID NO.5 of the sequence listing. tktA The nucleotide sequence of the gene is shown in SEQ ID NO.6 of the sequence listing. aroG fbr The nucleotide sequence of the gene is shown in SEQ ID NO.7 of the sequence listing. tyrA fbr The nucleotide sequence of the gene is shown in SEQ ID NO.8 of the sequence listing. pgm The nucleotide sequence of the gene is shown in SEQ ID NO.9 of the sequence listing. galU The nucleotide sequence of the gene is shown in SEQ ID NO.10 of the sequence listing. gntK The nucleotide sequence of the gene is shown in SEQ ID NO.11 of the sequence listing.

5. The rhodioloside-producing strain according to claim 2, characterized in that: The nucleotide sequence of the trc promoter is shown in SEQ ID NO.18, and the nucleotide sequence of the lac promoter is shown in SEQ ID NO.

19.

6. The method for constructing the rhodioloside-producing strain according to any one of claims 1-5, characterized in that: In the originating strain E. coli The following are the specific steps for targeted modification based on the W3110: (1) Knockout of repressor regulatory protein genes on the genome tyrR ; (2) Enhancement of Saccharomyces cerevisiae-derived yeast using the TRC promoter ARO10 Gene; (3) Enhancement of Saccharomyces cerevisiae-derived yeast using the TRC promoter ADH6 Gene; (4) Enhance the osmanthus-derived osmanthus source using the trc promoter. Oft8gt1 Genes, on Oft8gt1 The gene is copied twice; (5) Knockout of the gene encoding UDP-glucose hydrolase in the genome ushA Genes and pyruvate phosphotransferase pykF Gene; (6) Enhancement using the trc promoter tktA Gene transcription; (7) The gene encoding hydroxycarboxylic acid dehydrogenase was knocked out in the genome. ldhA Genes and encoding phenylacetaldehyde dehydrogenase feaB Gene; (8) Enhancement using the trc promoter aroG fbr Gene transcription, on aroG fbr The gene is copied twice; (9) Strengthening using the trc promoter tyrA fbr Gene transcription; (10) Utilizing the lac promoter to enhance the efficacy of Lactobacillus brevis-derived probiotics TDC Gene transcription; (11) Using the lac promoter to enhance the efficacy of Micrococcus luteus-derived probiotics TYO Gene; (12) Enhancement using the trc promoter gntK Gene; (13) Using the lac promoter to enhance Bacillus-derived [products / products] GDH Gene; (14) Enhancement using the trc promoter pgm Gene; (15) Enhancement using the trc promoter galU Gene.

7. The use of the rhodioloside-producing strain according to any one of claims 1-5 in the production of rhodioloside.

8. The application according to claim 7, characterized in that: Rhodioloside is produced by fermentation in shake flasks or fermenters.

9. The application according to claim 7 or 8, characterized in that: The specific steps are as follows: (1) Slant culture: The rhodioloside-producing strain was inoculated onto a slant culture medium and cultured at 36-37℃ for 12-16h; (2) Shake flask seed culture: Take solid slant inoculum and inoculate it into shake flask culture medium for fermentation. The culture temperature is 36-37℃, the culture time is 12-20h, the shaking speed is 200-240r / min, and the pH is 6.4-6.

7. (3) Shake flask fermentation culture: the fermentation inoculum amount is 1-5%, the culture temperature is 32-34℃, the pH is 6.4-6.7, the culture time is 24-36h, and the shaking speed is 180-220r / min.

10. The application according to claim 9, characterized in that: The slant culture medium used in step (1) is general-purpose LB solid medium; the seed culture medium used in step (2) is: glucose 20-30 g / L, glycerol 20-30 g / L, yeast extract powder 3-5 g / L, peptone 1.2-1.5 g / L, anhydrous magnesium sulfate 1-2 g / L, potassium dihydrogen phosphate 1-3 g / L, ammonium sulfate 1-2 g / L, ferrous sulfate heptahydrate 20-40 mg / L, manganese sulfate monohydrate 10- 20 mg / L, the remainder being water; the fermentation medium used in step (3) is: yeast extract powder 4-5 g / L, peptone 2-3 g / L, corn steep liquor 3 g / L, anhydrous magnesium sulfate 1.5-2 g / L, potassium dihydrogen phosphate 1-3 g / L, ammonium sulfate 1-2 g / L, ferrous sulfate heptahydrate 20-40 mg / L, manganese sulfate monohydrate 10-20 mg / L, VB1, VB3, and VB5 each 2-3 mg / L, the remainder being water.

Images