Construction, production method and application of genetically engineered bacterium for synthesizing Gadusol

Abstract

The invention relates to a construction method, a production method and application of a genetically engineered bacterium for synthesizing Gadusol, the genetically engineered bacterium for synthesizing Gadusol is obtained by taking escherichia coli MG1655 as an initial strain, knocking out a ketoaldolase gene on an escherichia coli MG1655 genome and cooperatively expressing an exogenous EEVS gene and an exogenous MT-Ox gene. According to the invention, an EEVS gene and a DrMT-Ox gene are integrated to a specific gene locus of an escherichia coli chromosome in a multi-copy form, and a key enzyme gene of a phosphate pentose pathway (PPP) is integrated and overexpressed on the chromosome, so that efficient biosynthesis of Gadusol is realized in escherichia coli for the first time; the method does not need exogenous plasmids, does not depend on inducers and antibiotics, can efficiently utilize a cheap carbon source, and realizes high-yield, stable and green production of Gadusol.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and fermentation technology, specifically relating to the construction, production method and application of genetically engineered bacteria for synthesizing Gadusol. Background Technology

[0002] Gadusol is a cyclohexenone compound naturally found in various aquatic organisms, such as zebrafish, sea urchin eggs, and artichokes. Despite its simple molecular structure, it possesses strong ultraviolet absorption capabilities (especially in the UVB / UVC band) and significant antioxidant activity, thus showing broad application prospects in high-end cosmetics, functional foods, and pharmaceutical preparations. Studies have shown that Gadusol can not only be used directly as a photoprotectant to mitigate UV-induced DNA damage, but also as a precursor for the synthesis of a series of UVA-protective spore-like amino acids (MAAs), further expanding its application scope.

[0003] Currently, Gadusol production primarily relies on extraction from marine biological tissues. However, its content in organisms is extremely low (typically found in trace amounts in specific tissues such as fish eggs and embryos), and the extraction process is complex, costly, and puts pressure on marine resources, making it difficult to meet the demands for large-scale, sustainable production. Regarding microbial synthesis, studies have attempted to construct Gadusol synthetic pathways in yeast systems. For example, through heterologous expression of zebrafish-derived... EEVS and MT-Ox Genes, and in Saccharomyces cerevisiae or Pichia pastoris ( Komagataella phaffii By knocking out the competitive transaldolase gene in [a specific culture medium], de novo synthesis of Gadusol was achieved. However, the reported yields remain low, with a maximum of 315.5 mg / L in shake-flask culture, and large-scale fermentation validation has yet to be observed. More importantly, these systems mostly rely on plasmid expression of exogenous genes, requiring continuous antibiotic addition to maintain plasmid stability and the use of inducers such as IPTG to initiate gene expression. This not only increases production costs but also introduces potential risks of drug-resistant gene diffusion and inducer toxicity, limiting the feasibility of their industrial application.

[0004] Therefore, developing a recombinant E. coli system that does not require exogenous plasmids, does not rely on inducers and antibiotics, and can efficiently utilize inexpensive carbon sources to achieve high-yield, stable, and green manufacturing of Gadusol has become a pressing technical challenge in this field. Summary of the Invention

[0005] The purpose of this invention is to overcome the defects in the prior art and to provide the construction, production method and application of genetically engineered bacteria for synthesizing Gadusol. This invention not only greatly increases the yield of Gadusol, but also eliminates the need for exogenous plasmids, does not rely on inducers and antibiotics, and can efficiently utilize inexpensive carbon sources, thus achieving high-yield, stable and green manufacturing of Gadusol.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A genetically engineered bacterium that synthesizes Gadusol uses Escherichia coli MG1655 as the initial strain, knocks out the transketolase gene in the E. coli MG1655 genome, and co-expresses exogenous Gadusol. EEVS Genes and exogenous MT-Ox Gene acquisition.

[0007] As a further technical solution, the transketaldase gene includes the transketaldase A gene. talA and transketaldase B gene talB .

[0008] As a further technical solution, the external source EEVS Genes derived from zebrafish EEVS Gene (gene ID. LOC100003999, its nucleotide sequence is shown in SEQ ID NO.1); As a further technical solution, the external source MT-Ox Genes derived from zebrafish MT-Ox Gene (gene ID. zgc:113054, its nucleotide sequence is shown in SEQ ID NO.3).

[0009] As a further technical solution, exogenous... EEVS Genes and exogenous MT-Ox Genes are generated by promoter P J23119 Start the expression.

[0010] As a further technical solution, knocking out the gene of E. coli MG1655... nagB Gene, gcd Gene 、edd Genes, and in nagB Gene, gcd Gene 、edd Exogenous genes are integrated at specific sites. EEVS Gene.

[0011] As a further technical solution nagB Gene: Annotation: NC_000913.3(702811..703611, complement).

[0012] As a further technical solution gcd Gene: Annotation: NC_000913.3(138835..141225, complement).

[0013] As a further technical solution edd Gene: Annotation: NC_000913.3(1932793..1934604, complement).

[0014] As a further technical solution, knocking out the gene of E. coli MG1655... arsB Gene 、ybeQ Gene 、 purR Genes, and in arsB Gene 、ybeQ Gene 、purR Exogenous genes are integrated at specific sites. MT-Ox Gene.

[0015] As a further technical solution arsB Gene: Annotation: NC_000913.3(3648935..3650224).

[0016] As a further technical solution ybeQ Gene: Annotation: NC_000913.3(675570..676547, complement).

[0017] As a further technical solution purR Gene: Annotation: NC_000913.3(1737844..1738869).

[0018] As a further technical solution, a genetically engineered bacterium for synthesizing Gadusol uses Escherichia coli MG1655 as the initial strain and also knocks out... ldhA Genes, and in ldhA The gene locus integrates the key enzyme gene of the pentose phosphate pathway (PPP).

[0019] As a further technical solution, the key enzyme genes of the pentose phosphate pathway (PPP) include pgl Gene, zwf Gene 、gnd Gene 、rpiA Gene 、rpe Gene 、tktA Gene 、tktB One or more of the genes (preferably) rpiA Gene).

[0020] As a further technical solution, the key enzyme gene of the pentose phosphate pathway (PPP) is promoted using the promoter P. J23119 Start the expression.

[0021] As a further technical solution ldhA Gene: Annotation: NC_000913.3(1441854..1442843, complement).

[0022] As a further technical solution pgl Gene: Annotation: NC_000913.3(798586..799581).

[0023] As a further technical solution zwf Gene: Annotation: NC_000913.3(1934839..1936314, complement).

[0024] As a further technical solution gnd Gene: Annotation: NC_000913.3(2099862..2101268, complement).

[0025] As a further technical solution rpiA Gene: Annotation: NC_000913.3(3058666..3059325, complement).

[0026] rpe Gene: Annotation: NC_000913.3 (3514382..3515059, complement).

[0027] As a further technical solution, the promoter P is adopted. J23119 Start Expression pgl Gene, zwf Gene 、gnd Gene 、 rpiA Gene 、rpe Gene 、tktA Gene 或tktB Gene.

[0028] A genetically engineered bacterium that synthesizes Gadusol, using Escherichia coli MG1655 as the initial strain, underwent the following modifications (A and B): A: Knockout of the transketaldase A gene in the genome of E. coli MG1655 talAand transketaldase B gene talB ; B: Knockout of the E. coli MG1655 genome nagB Gene, gcd Gene 、edd Genes, and in nagB Gene, gcd Gene 、edd Integrating at the gene locus EEVS Gene; Knockout of the genome of E. coli MG1655 arsB Gene 、ybeQ Gene 、purR Genes, and in arsB Gene 、 ybeQ Gene 、purR Integrating at the gene locus MT-Ox Gene.

[0029] [[ID=7 Genes and ​ Genes are generated by promoter P J23119 Start the expression.

[0030] C: Knockout ​ Genes, and in ​ The gene locus integrates a key enzyme gene of the pentose phosphate pathway (PPP) from the genome of *E. coli* MG1655; the key enzyme gene of the pentose phosphate pathway (PPP) is promoted using the P... J23119 Start the expression.

[0031] The method for producing Gadusol using the genetically engineered bacteria involves inoculating the genetically engineered bacteria into a fermentation medium, using glycerol as the carbon source and xylose as the substrate, and fermenting to produce Gadusol.

[0032] Application of the genetically engineered bacteria in the fermentation production of Gadusol.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The present invention will include the aforementioned ​ Genes and ​ Genes are integrated into specific loci on the E. coli chromosome in multiple copy form (e.g. ​ (etc.), and use strong constitutive promoters to initiate their expression, thereby constructing stable engineered strains that do not require exogenous plasmids, antibiotics and inducers; 2. High-yield Gadusol: This is achieved through further integration and overexpression of key enzyme genes in the pentose phosphate pathway (PPP) on the chromosome (such as...). ​ (etc.), to enhance the supply of the precursor sedoheptulose-7-phosphate (S7P), ultimately achieving a significant increase in Gadusol production.

[0034] In summary, the recombinant Escherichia coli system constructed in this invention has achieved the first efficient biosynthesis of Gadusol in Escherichia coli, and has achieved the highest yield reported to date: a yield of 668.09 mg / L in shake flask fermentation and a yield of 2.43 g / L in a 5L feed-by-batch fermenter, which is significantly higher than that of engineered strains in existing yeast production systems. Moreover, it does not require exogenous plasmids, does not rely on inducers and antibiotics, and can efficiently utilize inexpensive carbon sources, thus achieving high-yield, stable, and green manufacturing of Gadusol. Attached Figure Description

[0035] ​ A comparison of Gadusol yields for recombinant engineered strains MGG-01–MGG-08; ​ A comparison of Gadusol yields for recombinant engineered strains MGG-09–MGG-13; ​ A comparison of Gadusol yields for recombinant engineered strains MGG-13A—MGG-13G. Detailed Implementation

[0036] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] Additionally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0038] In this invention, the plasmids, restriction enzymes, PCR enzymes, column-based DNA extraction kits, and DNA gel recovery kits used in the following examples were all commercially available products, and the specific operations were performed according to the kit instructions. Conventional procedures such as colony PCR, nucleic acid agarose gel electrophoresis, heat shock transformation, electroporation, preparation of competent cells, and extraction and preservation of bacterial genomes were performed according to *Molecular Cloning: A Laboratory Manual (Fourth Edition)*. Sequencing of the plasmids and DNA products was performed by Genewiz (Suzhou).

[0039] (a) Culture medium (1) LB liquid medium: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L.

[0040] (2) LB solid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar powder.

[0041] (3) Fermentation medium: 15 g / L glycerol, 15 g / L xylose, 10.0 g / L yeast extract, 3.0 g / L ammonium sulfate, 2.0 g / L diammonium hydrogen phosphate, 6.75 g / L potassium dihydrogen phosphate, 0.7 g / L magnesium sulfate heptahydrate, 10 g / L ferrous sulfate heptahydrate, 2.25 g / L zinc sulfate heptahydrate, 1 g / L copper sulfate pentahydrate, 0.5 g / L manganese sulfate monohydrate, 0.23 g / L sodium borate decahydrate, 2 g / L anhydrous calcium chloride, 0.1 g / L ammonium heptamolybdate.

[0042] (4) Fermentation tank culture medium: 30 g / L glycerol, 10.0 g / L yeast extract, 3.0 g / L ammonium sulfate, 2.0 g / L diammonium hydrogen phosphate, 6.75 g / L potassium dihydrogen phosphate, 0.7 g / L magnesium sulfate heptahydrate, 10 g / L ferrous sulfate heptahydrate, 2.25 g / L zinc sulfate heptahydrate, 1 g / L copper sulfate pentahydrate, 0.5 g / L manganese sulfate monohydrate, 0.23 g / L sodium borate decahydrate, 2 g / L anhydrous calcium chloride, and 0.1 g / L ammonium heptamolybdate.

[0043] (5) Feeding solution for batch fermentation: 600 g / L glycerol containing 20 g / L magnesium sulfate heptahydrate and 0.2 g / L thiamine, and 500 g / L xylose. pH adjustment: 14% ammonia water (v / v).

[0044] (II) Gadusol detection: Take 1 mL of fermentation broth, centrifuge at 10,000 rpm for 10 min, collect the supernatant, and use it for HPLC determination.

[0045] HPLC detection conditions: High performance liquid chromatography (HPLC) system (Agilent); Column: ZORBAX Eclipse Plus C18; Detector: Agilent UV detector; Mobile phase: 0.25% formic acid in water; Flow rate: 0.65 mL / min; Column temperature: 30 ℃; Injection volume: 10 μL.

[0046] (iii) Strains The dual plasmid gene editing system pEcCpf1 / pcrEG has been published in the literature: Zhu X, Wu Y, Lv X. Combining CRISPR–Cpf1 and Recombineering Facilitates Fast and Efficient Genome Editing in ​ [J]. ACS Synthetic Biology, 2022(5):11. The strains involved in the following examples are shown in Table 1; Table 1. Strains involved in the following examples

[0047] (iv) Primers: The primers required in the following examples are shown in Table 2; Table 2 Primers required in the following examples

[0048] (v) Unless otherwise specified, all raw materials used are commercially available.

[0049] Example 1: Construction of Gadusol synthase plasmid 1. Construction of pET-DrEEVS-DrMT-Ox and pET-ShEEVS-DrMT-Ox expression vectors The optimized codons are derived from zebrafish ( ​ )of ​ Gene (SEQ ID NO.1) and derived from Streptomyces hydrophila ( ​ )of ​ The gene (SEQ ID NO.2) was constructed into the first position of the multiple cloning site of the expression vector pET-PJ23119-PJ23119 to form recombinant plasmids pET-DrEEVS and pET-ShEEVS; The optimized codons are combined with those derived from zebrafish ( ​ )of ​ The gene (SEQ ID NO.3) was constructed into the multiple cloning site 2 of the expression vectors pET-DrEEVS and pET-ShEEVS to form recombinant plasmids pET-DrEEVS-DrMT-Ox and pET-ShEEVS-DrMT-Ox.

[0050] The specific steps are as follows: (1) PCR system (100 μl as an example): H2O: 45 μL; template: 0.5-1 μL; primer 1: 2 μL; primer 2: 2 μL; PrimerSTAR Max: 50 μl.

[0051] (2) Based on the PCR system in (1), the target gene is amplified using the corresponding primers, and the vector fragment is amplified using the plasmid to be constructed as a template using the corresponding primers.

[0052] (3) Verify the fragments amplified in (2) by nucleic acid electrophoresis and recover them using a kit.

[0053] (4) The fragments obtained in (3) are assembled using seamless cloning. The formula for calculating the amount of each fragment added is V = (0.02 × fragment length) / fragment concentration. The reaction is carried out at 50 °C for 5 minutes for ligation.

[0054] (5) The assembled plasmid is transferred into the cloning host, plated on the corresponding LB plate and cultured overnight at 37 °C. Then, a single colony is picked and the plasmid is extracted for Sanger sequencing. Successful sequencing completes the construction of the recombinant plasmid.

[0055] (6) The PCR conditions, gel electrophoresis, gel recovery and purification and seamless cloning operations constructed in the second step are the same as above. After sequence comparison, the recombinant plasmids pET-DrEEVS-DrMT-Ox and pET-ShEEVS-DrMT-Ox were finally obtained.

[0056] Example 2: Construction of Gadusol chassis strain Knockout of the transketaldase A gene in the E. coli genome using the CRISPR / Cpf1 gene editing system ​ and transketaldase B gene ​ strain MGG-A was obtained. 、 MGG-B and MGG-AB; for CRISPR / Cpf1 gene editing systems, please refer to the reference Zhu X, Wu Y, Lv X. Combining CRISPR–Cpf1 and Recombineering Facilitates Fast and Efficient Genome Editing in ​ [J]. ACS SyntheticBiology, 2022(5):11. 1. Genes ​ , ​ The knockout method is the same, with ​ For example, let's introduce genes.​ The specific steps of genome knockout are as follows: (1) Based on the transketolase A gene that needs to be knocked out in the host bacteria ​ Primers talA-UH-F / R and talB-UH-F / R were designed to amplify homologous arms of 500 bp upstream and downstream of talA, and the two fragments were ligated in an overlapping manner to serve as donor DNA fragments (Table 2).

[0057] (2) Using the original commercial plasmid pcrEG as a template, the N plasmid on pcrEG was amplified by PCR using primers talA-N23-F / R. 23 Sequence replacement with ​ complementary N 23 Sequences, obtaining targeted sequences ​ pcrEG plasmid pcrEG- ​ The PCR product was used to remove template DNA using DpnI enzyme, and then transformed into E. coli JM109 competent cells using heat shock transformation. The cells were then plated on LB agar plates containing spectinomycin and cultured at 37 °C to extract plasmids and sequence them.

[0058] (3) Take pEcCpf1 plasmid and transform it into Escherichia coli MG1655 electrocompetent cells. Spread the transformed bacterial solution onto LB agar containing kanamycin and incubate overnight at 37 ℃. Inoculate the single colonies that grow above into test tubes containing 4 mL of LB medium containing kanamycin and incubate for 12 h to prepare electrocompetent cells.

[0059] (4) Place electrocompetent cells containing pEcCpf1 plasmid on ice, thaw, and then add pcrEG- ​ 200 ng of plasmid and 800 ng of donor DNA fragment were mixed and transferred to a pre-chilled 2 mm electroporation cuvette and incubated on ice. The cuvette was placed in an electroporator and electroporated at 2.5 kV for approximately 0.5 ms. Immediately afterward, 1 mL of pre-chilled LB medium was added, and the mixture was incubated at 37 °C and 200 r / min for 2 h. Subsequently, the mixture was centrifuged at 3,500 rpm for 3 min, the supernatant was discarded, and the bacterial cells were thoroughly mixed and spread onto double-antibiotic plates (final concentrations of 50 μg / L kanamycin and 50 μg / L spectinomycin). The plates were then incubated upside down at 37 °C for 12–24 h.

[0060] (5) The colonies were sent to Suzhou Genewiz Biotechnology Co., Ltd. for sequencing. The above positive clones were picked into 4 mL LB liquid tubes (containing 10 mM rhamnose and 4 μL Kan), and incubated at 37℃ for 12 h to remove pcrEG- ​Plasmid removal. The successfully removed recombinant strain was then transferred to a 4 mL LB liquid tube (containing a final concentration of 5 g / L glucose) and incubated at 37°C for 12 h to remove the pEcCpf1 plasmid. Successful removal of the pEcCpf1 plasmid yielded recombinant *E. coli* MG1655Δ. ​ Engineered bacteria (i.e., MGG-A).

[0061] (6) Other knockout procedures are the same as above, resulting in MGG-B and MGG-AB.

[0062] 2. Integration of key heterologous enzyme genes and key precursor enzyme genes Using MGG-AB as the host strain, knockout ​ Genes and ​ Gene locus integration ​ Gene knockout ​ Genes and ​ Gene locus integration ​ Gene, obtain MGG-ABΔ ​ ::P J23119 - ​ Δ ​ ::P 23119 - ​ (MGG-09); Using MGG-09 as the host strain, knockout ​ Genes and ​ Gene locus integration ​ Gene, obtain MGG-09Δ ​ :: P J23119 - ​ (MGG-10); Using MGG-10 as the host strain, knockout ​ Genes and ​ Gene locus integration ​ Gene, obtain MGG-10Δ ​ :: P J23119 - ​ (i.e., MGG-11); Using MGG-11 as the host strain, knockout ​ Genes and ​ Gene locus integration ​ Gene, obtain MGG-11Δ ​ :: P J23119 - ​ (MGG-12); Using MGG-12 as the host strain, knockout ​ Genes and ​ Gene locus integration ​ Gene, obtain MGG-12Δ ​ :: PJ23119 - ​ (MGG-13); Using MGG-13 as the host strain, knockout ​ Genes and ​ Gene loci are integrated separately ​ Gene, ​ Gene ​ Gene ​ Gene ​ Gene ​ Gene ​ Gene; Obtain MGG-13Δ ​ ::P J23119 - ​ (MGG-13A) 、 MGG-13Δ ​ ::P J23119 - ​ (MGG-13B) 、 MGG-13Δ ​ ::P J23119 - ​ (MGG-13C) 、 MGG-13Δ ​ ::P J23119 - ​ (MGG-13D) 、 MGG-13Δ ​ ::P J23119 - ​ (MGG-13E) 、 MGG-13Δ ​ ::P J23119 - ​ (MGG-13F) 、 MGG-13Δ ​ ::P J23119 - ​ (MGG-13G) 。

[0063] Among them, knockout ​ Genes and ​ Gene locus integration ​ Taking genes as an example, the integration process of key heterologous enzyme genes and key precursor enzyme genes is introduced as follows: (1) The procedure for integrating genes is the same as that for knocking out genes. ​ For example, based on the host bacteria that need to be knocked out ​ The gene was amplified by PCR using the *E. coli* MG1655 genome as a template and nagB-UH-F / R and nagB-DH-F / R primers (Table 2). ​Upstream and downstream fragments homologously complementary near the gene were purified by gel electrophoresis and gel recovery. Then... ​ Using the gene as a template, primers DrEEVS-F / R (Table 2) were used to amplify the gene via PCR. ​ The gene fragments were then purified by gel electrophoresis and gel recovery. The three gene fragments were then fused using an overlap PCR method to obtain a complete template gene fragment, which was used as the donor DNA fragment.

[0064] (2) For the remaining experimental procedures, refer to the gene knockout procedure.

[0065] (3) Other integration processes are the same as above, and strains MGG-09—MGG-13 and MGG-13A—MGG-13G are finally obtained.

[0066] Example 3: Obtaining engineered strains by heat shock transformation The pET-DrEEVS-DrMT-Ox and pET-ShEEVS-DrMT-Ox constructed in Example 1 were transformed into chemocompetent cells (strains MGG and MGG-A) respectively using the heat shock transformation method. 、 The fermentation engineered bacteria MGG-01—MGG-08 were obtained from MGG-B and MGG-AB. The specific steps are as follows: (1) The amount of plasmid added to the competent cells should not exceed 1 / 10 of the competent cell volume (generally 5 μL is added to 100 μL of competent cells). After adding the plasmid, shake it well with the pipette tip.

[0067] (2) After adding plasmid, competent cells were placed in an ice bath for 20 min and then heat-shocked at 42 °C for 90 s.

[0068] (3) After 5 min in an ice bath, add 900 μL of antibiotic-free LB medium and incubate in a shaker at 37 °C for 1 h.

[0069] (4) After enriching the bacterial cells by centrifuging at 5,000 rpm for 5 min in a centrifuge, the cells were spread on solid plates containing the corresponding resistance and incubated overnight at 37 ℃ to obtain the fermentation strain.

[0070] Example 4: Shake Flask Fermentation Production The constructed plasmid-type fermentation engineered bacteria (MGG-01—MGG-08) and whole-genome-type fermentation engineered bacteria (MGG-09—MGG-13 and MGG-13A—MGG-13G) were inoculated into LB liquid medium and cultured overnight at 37 ℃ and 200 rpm for 12 h to obtain seed culture. 0.5 mL of the seed culture was inoculated into 25 mL of fermentation medium and cultured at 37 ℃ and 200 rpm until OD... 600The pH was set to 0.6–0.8, then the temperature was lowered to 25 °C, and the culture was continued at 200 rpm for 72 h. One mL of fermentation broth was centrifuged at 10,000 rpm for 10 min, and the supernatant was collected for HPLC analysis. The results are shown below. ​ ; The strain MGG-13D, which ultimately produced the highest yield in shake-flask culture, achieved a Gadusol yield of 668.09 mg / L, far exceeding that of the plasmid-type fermentation engineered strains (MGG-01—MGG-08).

[0071] Example 5: Fermentation tank fed-batch culture A fed-batch fermentation experiment was conducted on strain MGG-13D in a 5 L fermenter.

[0072] The strain MGG-13D was inoculated into 4 mL of antibiotic-free LB medium and cultured overnight at 37 ℃ and 200 rpm for 12 h to obtain primary seed culture. 1 mL of the primary seed culture was then inoculated into 100 mL of fermentation medium and cultured at 37 ℃ and 200 rpm until OD reached [value missing]. 600 The OD value was 1.5–2.0 to obtain a secondary seed culture. 200 mL of this secondary seed culture was inoculated into a 5L fermenter containing 2 L of fermenter medium for fermentation culture. The initial temperature was maintained at 37 °C. After the initial 30 g / L glycerol in the medium was completely depleted, a feed solution was added to maintain a final glycerol concentration of 10 g / L and a final xylose concentration of 10 g / L to meet cell growth requirements. When the OD value… 600 When the temperature reached 30 °C, it was slowly cooled to 25 °C, and xylose was added as a substrate. The pH was maintained at 6.8 ± 0.2 throughout the process, and foaming was controlled by adding an antifoaming agent. Dissolved oxygen was controlled by adjusting the stirring speed (100–900 rpm) and aeration rate (2–8 vvm). During fermentation, samples were taken every 5 hours to determine the content of substrate glycerol and xylose, as well as product Gadusol. The results are shown in Table 3. Table 3

[0073] Table 3 shows that strain MGG-13D achieved a Gadusol yield of 2.43 mg / L in a 5 L fermenter after 55 h, with an OD of [missing value]. 600 With a maximum value of 167.8, it demonstrates significant production potential in large-scale industrial applications.

[0074] The embodiments described above are merely preferred embodiments of the present invention, and not an exhaustive list of all possible implementations of the present invention. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. A genetically engineered bacterium that synthesizes Gadusol, characterized in that, Using Escherichia coli MG1655 as the initial strain, the transketolase gene in the E. coli MG1655 genome was knocked out and exogenous [genes / products] were co-expressed. EEVS Genes and exogenous MT-Ox Gene acquisition.

2. The genetically engineered bacterium for synthesizing Gadusol according to claim 1, characterized in that, The transketaldase gene includes the transketaldase A gene. talA and transketaldase B gene talB .

3. The genetically engineered bacterium for synthesizing Gadusol according to claim 1, characterized in that, The external source EEVS Genes derived from zebrafish EEVS Gene; The external source MT-Ox Genes derived from zebrafish MT-Ox Gene.

4. The genetically engineered bacterium for synthesizing Gadusol according to claim 1, characterized in that, exogenous EEVS Genes and exogenous MT-Ox Genes are generated by promoter P J23119 Start the expression.

5. The genetically engineered bacterium for synthesizing Gadusol according to claim 1, characterized in that, Co-expression of exogenous EEVS Genes and exogenous MT-Ox Genes, including: Knockout of the genome of E. coli MG1655 nagB Gene, gcd Gene edd Genes, and in nagB Gene, gcd Gene edd Exogenous genes are integrated at specific sites. EEVS Gene; Knockout of the genome of E. coli MG1655 arsB Gene ybeQ Gene purR Genes; and arsB Gene ybeQ Gene purR Exogenous genes are integrated at specific sites. MT-Ox Gene.

6. The genetically engineered bacterium for synthesizing Gadusol according to claim 1, characterized in that, Using Escherichia coli MG1655 as the initial strain, the following modifications were also performed: knockout ldhA Genes, and in ldhA The gene locus integrates key enzyme genes of the pentose phosphate pathway, including... pgl Gene, zwf Gene ,gnd Gene rpiA Gene 、 rpe Gene tktA Gene tktB One or more genes.

7. The genetically engineered bacterium for synthesizing Gadusol according to claim 6, characterized in that, Using promoter P J23119 The key enzyme gene that initiates the expression of the pentose phosphate pathway.

8. A method for producing Gadusol using the genetically engineered bacteria as described in any one of claims 1 to 6, characterized in that, The genetically engineered bacteria were inoculated into a fermentation medium and fermented to produce Gadusol using glycerol as a carbon source and xylose as a substrate.

9. The application of the genetically engineered bacteria as described in any one of claims 1 to 6 in the fermentation production of Gadusol.

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