Recombinant engineering bacterium for producing pyrone derivative as well as construction method and application of recombinant engineering bacterium

Abstract

The invention relates to a recombinant engineering bacterium for producing a pyrone derivative as well as a construction method and application of the recombinant engineering bacterium, and belongs to the technical field of biological engineering. By optimizing the metabolic flux of malonyl coenzyme A and 3-hydroxy propionyl coenzyme A, a novel pyrone derivative 4-hydroxy-6-hydroxyethyl-2-pyrone (HHEP) is obtained, a complete heterologous synthesis path of the pyrone derivative 4-hydroxy-6-hydroxyethyl-2-pyrone (HHEP) is constructed in recombinant engineering bacteria, efficient de novo synthesis of the HHEP is realized, the yield of the HHEP is increased, and the yield of the HHEP is increased. Glucose is used as a single carbon source, a toxic reagent or a complex purification process is not needed, and the shake flask yield of HHEP within 120 hours reaches 3.6 g / L. The HHEP has antibacterial activity, and a breakthrough technical scheme is provided for low-cost and sustainable manufacturing of a novel antibacterial agent. Meanwhile, the recombinant engineering bacterium provides a breakthrough technical scheme for low-cost and sustainable manufacturing of the HHEP.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a recombinant engineered bacterium for producing pyranone derivatives, its construction method, and its application. Background Technology

[0002] Pyranones are widely distributed in nature as secondary metabolites of plants and microorganisms, and are known to possess various biological activities such as antibacterial, antioxidant, and anti-inflammatory effects. However, traditional chemical synthesis of these oxygen-containing heterocyclic compounds usually relies on multi-step organic reactions, which suffer from harsh reaction conditions, low yields, and environmental pollution. Microbial synthesis, on the other hand, has emerged as an alternative due to its green and sustainable characteristics.

[0003] Yarrowia lipolytica offers significant advantages in metabolic programmability and industrial production. It effectively utilizes inexpensive substrates as carbon sources and exhibits strong tolerance and robustness, enabling it to grow under diverse environmental conditions. Furthermore, Yarrowia lipolytica demonstrates a marked advantage in heterologous gene expression; therefore, it is an ideal microbial platform. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a recombinant engineered bacterium for producing pyranone derivatives, its construction method, and its application.

[0005] This invention relates to metabolic engineering of *Yersinia lipolytica*, revealing that the modified recombinant strain can efficiently secrete a novel pyranone derivative. This pyranone derivative, identified as 4-hydroxy-6-hydroxyethyl-2-pyrone (HHEP), is an oxoheterocyclic compound containing both hydroxyl and hydroxyethyl functional groups, endowing it with unique chemical and biological activities. As a novel, non-natural pyranone derivative, HHEP has not previously been systematically characterized or functionally studied, and its natural source and synthetic route remain unclear. This invention provides a novel pyranone derivative, HHEP, and a method for de novo synthesis of HHEP based on metabolic engineering of *Yersinia lipolytica*, offering a breakthrough technical solution for the low-cost and sustainable manufacturing of HHEP.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a pyranone derivative, named 4-hydroxy-6-hydroxyethyl-2-pyrone (HHEP), with the structural formula shown in formula (I).

[0008]

[0009] The pyranone derivative HHEP of the present invention has the molecular formula C7H8O4 and the molecular weight 156.14.

[0010] The synthesis of HHEP in this invention is achieved by constructing a dual-pathway parallel-catalyzed malonyl-CoA biosynthetic pathway, a cascade enzyme-driven 3-hydroxypropionyl-CoA generation pathway, and an HHEP assembly module. Figure 11 In the malonyl-CoA biosynthesis pathway, aspartic acid is converted to β-alanine by aspartate decarboxylase (SfADC), which then undergoes a two-branched metabolism, either through direct oxidation by monoamine oxidase (EcMAO) or a transamination reaction mediated by endogenous β-alanine pyruvate aminotransferase (YlBAPAT), to generate malonyl hemialdehyde. Finally, it is reduced to malonyl-CoA by the C-terminus (CaMCR-C) of malonyl-CoA reductase. In the 3-hydroxypropionyl-CoA synthesis pathway, malonate... The semialdehyde is successively reduced to 3-hydroxypropionyl-CoA dehydrogenase (YlHBD1) to 3-hydroxypropionic acid, which is then activated by 3-hydroxyisobutyryl-CoA hydrolase (YlEHD3) to generate 3-hydroxypropionyl-CoA. In the HHEP assembly module, heterologous 2-pyranone synthase (Gh2PS) uses 3-hydroxypropionyl-CoA as the starting unit and undergoes condensation and cyclization with two molecules of malonyl-CoA (extension unit) to finally form 4-hydroxy-6-hydroxyethyl-2-pyranone (HHEP).

[0011] Secondly, the present invention provides the application of the 2-pyranone synthase (Gh2PS) encoding gene and the aspartate decarboxylase (SfADC) encoding gene in the preparation of recombinant engineered bacteria for producing the above-mentioned pyranone derivative (HHEP); the nucleotide sequence of the 2-pyranone synthase (Gh2PS) encoding gene is shown in SEQ ID NO:4; the nucleotide sequence of the aspartate decarboxylase (SfADC) encoding gene is shown in SEQ ID NO:1.

[0012] This invention has shown that by simultaneously expressing the 2-pyranone synthase (Gh2PS) encoding gene and the aspartate decarboxylase (SfADC) encoding gene in Yarrowia lipolytica Po1fk (Yarrowia lipolytica Po1f with the enzyme encoding gene ku70 responsible for non-homologous recombination knocked out), a recombinant engineered bacterium was constructed, which was able to ferment and produce 4-hydroxy-6-hydroxyethyl-2-pyranone (HHEP).

[0013] Thirdly, the present invention provides the application of the 2-pyranone synthase (Gh2PS) encoding gene, the aspartate decarboxylase (SfADC) encoding gene, the monoamine oxidase (EcMAO) encoding gene, and the malonyl-CoA reductase (CaMCR-C) encoding gene in the preparation of recombinant engineered bacteria for producing the above-mentioned pyranone derivative (HHEP); the nucleotide sequence of the 2-pyranone synthase (Gh2PS) encoding gene is shown in SEQ ID NO:4; the nucleotide sequence of the aspartate decarboxylase (SfADC) encoding gene is shown in SEQ ID NO:1; the nucleotide sequence of the monoamine oxidase (EcMAO) encoding gene is shown in SEQ ID NO:2; and the nucleotide sequence of the malonyl-CoA reductase (CaMCR-C) encoding gene is shown in SEQ ID NO:3.

[0014] The aspartate decarboxylase (SfADC) is derived from Shigella flexneri; the monoamine oxidase (EcMAO) is derived from Escherichia coli; the malonyl-CoA reductase (CaMCR-C) is derived from Chloroflexus aurantiacus; and the 2-pyranone synthase (Gh2PS) is derived from Gerbera hybrida.

[0015] This invention has found that by simultaneously expressing the gene encoding 2-pyranone synthase (Gh2PS) along with the genes encoding aspartate decarboxylase (SfADC), monoamine oxidase (EcMAO), and malonyl-CoA reductase (CaMCR-C) in Yarrowia lipolytica Po1fk to construct a recombinant engineered bacterium, the resulting recombinant engineered bacterium can also ferment and produce 4-hydroxy-6-hydroxyethyl-2-pyranone (HHEP), and the yield of HHEP is higher.

[0016] Fourthly, the present invention provides a recombinant expression vector comprising a 2-pyranone synthase (Gh2PS) gene expression cassette and an aspartate decarboxylase (SfADC) gene expression cassette.

[0017] The 2-pyranone synthase gene expression cassette includes a promoter, a Gh2PS encoding gene, and a terminator; the nucleotide sequence of the Gh2PS encoding gene is shown in SEQ ID NO:4;

[0018] The aspartate decarboxylase gene expression cassette includes a promoter, an SfADC encoding gene, and a terminator; the nucleotide sequence of the SfADC encoding gene is shown in SEQ ID NO:1.

[0019] This invention utilizes the 2-pyranone synthase gene expression cassette (P TEFin -Gh2PS-T Xpr2 The aspartate decarboxylase gene expression cassette (P) was introduced into Yarrowia lipolytica Po1f, i.e., Yarrowia lipolytica Po1fk, where the enzyme encoding gene ku70 responsible for non-homologous recombination was knocked out, resulting in recombinant strain 1. Then, the aspartate decarboxylase gene expression cassette (P) was introduced into recombinant strain 1. FBA -SfADC-T Pex20 Recombinant strain 3 was obtained, and HHEP could be produced by fermentation using glucose as the sole carbon source. This provides a recombinant expression vector for the production of HHEP using engineered bacteria.

[0020] As a preferred embodiment of the recombinant expression vector of the present invention, the recombinant expression vector further includes a monoamine oxidase (EcMAO) gene expression cassette and a malonyl-CoA reductase (CaMCR-C) gene expression cassette.

[0021] The monoamine oxidase gene expression cassette includes a promoter, an EcMAO encoding gene, and a terminator, and the nucleotide sequence of the EcMAO encoding gene is shown in SEQ ID No:2;

[0022] The malonyl-CoA reductase gene expression cassette includes a promoter, a CaMCR-C encoding gene, and a terminator; the nucleotide sequence of the CaMCR-C encoding gene is shown in SEQ ID No:3.

[0023] This invention utilizes the 2-pyranone synthase gene expression cassette (P TEFin -Gh2PS-T Xpr2 Recombinant strain 1 was obtained by introducing the Yarrowia lipolytica Po1f strain (i.e., Yarrowia lipolytica Po1fk) into recombinant strain 1, in which the enzyme encoding gene ku70 responsible for non-homologous recombination was knocked out. Then, the aspartate decarboxylase gene expression cassette (P) was introduced into recombinant strain 1. FBA -SfADC-T Pex20 Monoamine oxidase gene expression cassette (P TDH1 -EcMAO-T Mig1 ) and malonyl-CoA reductase gene expression cassette (P TEFin -CaMCR-CT Xpr2 Recombinant strain 2 was obtained. Using glucose as the single carbon source, HHEP was produced by fermentation using recombinant strain 2, and the yield was higher than that of recombinant strain 3.

[0024] In a preferred embodiment of the recombinant expression vector of the present invention, the promoter is P. TEFin P FBA or PTDH1 Any one of them, wherein the terminator is T Xpr2 T Pex20 or T Mig1 Any one of them.

[0025] In a preferred embodiment of the recombinant expression vector of the present invention, the recombinant expression vector further includes a vector.

[0026] As a preferred embodiment of the recombinant expression vector of the present invention, the vector includes plasmid pUrLp or plasmid pUC-HUH.

[0027] Fifthly, the present invention provides a recombinant engineered bacterium, wherein the above-mentioned recombinant expression vector is transferred into a host bacterium to construct the recombinant engineered bacterium.

[0028] The recombinant engineered bacteria (recombinant bacteria 2 and 3) containing the above recombinant expression vectors can produce HHEP.

[0029] The recombinant expression vector was introduced into the host bacterium *Yarrowia lipolytica* Po1fk in the form of a linearized plasmid, and then integrated into the genome via homologous recombination. The gene integration sites included: IntA, IntB, IntC1, IntC3, IntD, IntE, IntF, Dga1, Lip1, or Scp2 sites from *Yarrowia lipolytica* Po1fk. The recombinant expression vector was inserted between the upstream and downstream homologous sequences at the aforementioned sites.

[0030] In a preferred embodiment of the recombinant engineered bacteria of the present invention, the host bacteria is Escherichia coli or yeast.

[0031] As a preferred embodiment of the recombinant engineered bacteria of the present invention, the yeast includes Candida albicans, Pichia pastoris, Rhodotorula buergerianum, Saccharomyces cerevisiae, or Yersinia lipolytica.

[0032] In a preferred embodiment of the recombinant engineered bacteria described in this invention, the host bacteria is *Yarrowia lipolytica* Po1f. *Yarrowia lipolytica* has significant advantages in heterologous gene expression, enabling efficient integration of the HHEP biosynthetic pathway. Furthermore, *Yarrowia lipolytica* exhibits excellent HHEP secretion capabilities, which helps simplify the downstream extraction process and reduce separation costs. Therefore, *Yarrowia lipolytica* is an ideal microbial platform for HHEP production.

[0033] In a sixth aspect, the present invention provides a method for producing the above-mentioned pyranone derivative (HHEP), wherein the above-mentioned recombinant engineered bacteria are inoculated into a fermentation medium for fermentation culture to obtain a fermentation product containing 4-hydroxy-6-hydroxyethyl-2-pyranone; the fermentation medium is formulated as follows: water, glucose 20-80 g / L, yeast extract 1-10 g / L and peptone 2-20 g / L.

[0034] As a preferred embodiment of the method described in this invention, the fermentation culture medium is formulated as follows: water, glucose 20-80 g / L, yeast extract 1-10 g / L, peptone 2-20 g / L and aspartic acid 10-1000 mM.

[0035] As a preferred embodiment of the method described in this invention, the fermentation culture conditions are: 28-32℃, 200-250rpm shaking culture for 3-10 days.

[0036] In a preferred embodiment of the method described in this invention, the recombinant engineered bacteria are inoculated into a fermentation medium such that the initial OD600 is not greater than 2.

[0037] In a preferred embodiment of the method described in this invention, the recombinant engineered bacteria are prepared into a seed culture and then inoculated. The seed culture is prepared by culturing the activated recombinant engineered bacteria in YPD liquid medium at 30°C and 250 rpm for 16 hours to obtain the seed culture.

[0038] As a preferred embodiment of the method described in this invention, the above-mentioned fermentation product is resuspended in an aqueous solution containing grinding particles, freeze-ground, and centrifuged to obtain a supernatant containing 4-hydroxy-6-hydroxyethyl-2-pyranone.

[0039] In a seventh aspect, the present invention provides a fermentation product prepared using the above-described preparation method.

[0040] Eighthly, the present invention provides the use of the fermentation product as an antibacterial agent.

[0041] In a ninth aspect, the present invention provides the use of the above-mentioned pyranone derivative (HHEP) as an antibacterial agent.

[0042] This invention has found that fermentation products containing the novel pyranone derivative 4-hydroxy-6-hydroxyethyl-2-pyranone (HHEP) have significant antibacterial effects and can inhibit the growth of microorganisms. In YPD medium, the growth of HHEP engineered bacteria is significantly inhibited compared with the parent strain before modification.

[0043] In a tenth aspect, the present invention provides an antibacterial agent comprising the above-mentioned fermentation product or the above-mentioned pyranone derivative (HHEP).

[0044] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0045] This invention optimizes the metabolic flux of malonyl-CoA and 3-hydroxypropionyl-CoA to obtain a novel pyranone derivative, 4-hydroxy-6-hydroxyethyl-2-pyranone (HHEP). A complete heterologous synthetic pathway for HHEP was constructed in recombinant engineered bacteria, achieving efficient de novo synthesis using glucose as a single carbon source. No toxic reagents or complex purification processes are required, and the shake-flask yield of HHEP reaches 3.6 g / L within 120 hours. The HHEP of this invention exhibits antibacterial activity, providing a breakthrough technical solution for the low-cost and sustainable manufacturing of novel antibacterial agents. Furthermore, the recombinant engineered bacteria of this invention provide a breakthrough technical solution for the low-cost and sustainable manufacturing of HHEP. Attached Figure Description

[0046] Figure 1 This is the plasmid map of the initial plasmid pYLXP' in Example 1 of the present invention;

[0047] Figure 2 This is the plasmid map of pUrLp-ΔDga1 in Example 1 of the present invention;

[0048] Figure 3 This is the plasmid map of the recombinant plasmid pUrLp-ΔDga1-(CaMCR-C)-SfADC-EcMAO in Example 1 of the present invention;

[0049] Figure 4 This is the plasmid map of the recombinant plasmid pUrLp-ΔLip1-Gh2PS in Example 1 of the present invention;

[0050] Figure 5 This is the plasmid map of the recombinant plasmid pUrLp-ΔDga1-SfADC in Example 1 of the present invention;

[0051] Figure 6 This is the HPLC chromatogram of the supernatant in Example 3 of the present invention;

[0052] Figure 7 This is a comparison diagram of the synthesis of TAL and HHEP in Embodiment 3 of the present invention;

[0053] Figure 8 The LCMS spectrum of TAL in Embodiment 3 of the present invention;

[0054] Figure 9 This is the main peak mass spectrum (LCMS spectrum of HHEP) in Example 3 of the present invention;

[0055] Figure 10 This refers to the mass spectrometry fragment information of HHEP in Embodiment 3 of the present invention;

[0056] Figure 11 This is a diagram of the de novo synthesis path of HHEP in Embodiment 3 of the present invention;

[0057] Figure 12 The qualitative analysis results are those of the recombinant bacteria fermentation for HHEP production in Example 4 of this invention;

[0058] Figure 13 This is the quantitative analysis result of HHEP production by recombinant bacteria fermentation in Example 4 of the present invention;

[0059] Figure 14 This is a comparison of the growth curves of the strain in Example 5 of the present invention in different culture media. Detailed Implementation

[0060] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0061] Unless otherwise specified, all other materials and reagents used in the examples are commercially available.

[0062] Yarrowia lipolytica Po1f was purchased from the U.S. Culture Collection (ATCCMYA-2613). Yarrowia lipolytica Po1fΔku70 (MatA,Δku70,leu2-270,ura3-302,xpr2-322,axp1-2), abbreviated as Yarrowia lipolytica Po1fk, was constructed by knocking out the ku70 gene (GenBank accession number: YALI0C08701g) of Yarrowia lipolytica Po1f (published in Kretzschmar A, et al., Current Genetics, 2013, 59(1-2):63-72).

[0063] 2×Phanta Max Master Mix (Dye Plus), purchased from Nanjing Novozymes Biotechnology Co., Ltd.

[0064] Screening medium CSM-Ura: water, glucose 20 g / L, YNB (amino-free yeast nitrogen source) 6.7 g / L, CSM-Ura (complete supplement mixture to remove uracil) 0.67 g / L, agar powder 23 g / L.

[0065] Screening medium CSM-Leu: water, glucose 20 g / L, YNB (amino-free yeast nitrogen source) 6.7 g / L, CSM-Leu (complete supplement mixture with leucine removed) 0.67 g / L, agar powder 23 g / L.

[0066] YPD plate containing 5-fluoroorotic acid: water, containing 1 g / L 5-fluoroorotic acid, 20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose and 23 g / L agar powder.

[0067] Example 1

[0068] 1. Preparation of target gene

[0069] Based on the nucleotide sequences of the SfADC, EcMAO, CaMCR-C, and Gh2PS encoding genes (UniProt IDs: Q0T870, P46883, Q6QQP7, P48391) provided on UniProt, and after codon optimization, Suzhou Genewise Biotechnology Co., Ltd. was commissioned to synthesize the optimized SfADC, EcMAO, CaMCR-C, and Gh2PS encoding genes. The nucleotide sequence of SfADC is shown in SEQ ID No:1, the nucleotide sequence of EcMAO is shown in SEQ ID No:2, the nucleotide sequence of CaMCR-C is shown in SEQ ID No:3, and the nucleotide sequence of Gh2PS is shown in SEQ ID No:4.

[0070] Using Yarrowia lipolytica Po1fk genomic DNA as a template, promoter P was amplified. TEFin P TDH1 P FBA Sequence and Terminator T Xpr2 T Mig1 T Pex20 Sequence, P TEFin P TDH1 P FBA T Xpr2 T Mig1 T Pex20 The nucleotide sequences are shown in SEQ ID NO:5-10.

[0071] 2. Construction of single-gene recombinant plasmids

[0072] First, recombinant plasmids pYLXP'-Gh2PS, pYL31-SfADC, pYL24-EcMAO, and pYLXP'-(CaMCR-C) were constructed. Then, recombinant plasmids pUrLp-ΔLip1-Gh2PS and pUrLp-ΔDga1-(CaMCR-C)-SfADC-EcMAO were constructed.

[0073] The recombinant plasmids and their uses are shown in Table 1 below, and the primers used to construct the recombinant plasmids are shown in Table 2 below.

[0074] Table 1. Recombinant plasmids and their uses

[0075]

[0076] Table 2. Primer information used for constructing recombinant plasmids.

[0077]

[0078]

[0079] (1) Construction of recombinant plasmid pYLXP'-Gh2PS

[0080] The recombinant plasmid pYLXP'-Gh2PS uses the initial plasmid pYLXP' as a backbone, with the Gh2PS gene inserted into the promoter P of plasmid pYLXP'. TEFin and Termination T Xpr2 Between these, the Gh2PS gene expression cassette (P) is assembled. TEFin -Gh2PS-T Xpr2 ).

[0081] The initial plasmid pYLXP' contains the endogenous promoter P of Yarrowia lipolytica. TEFin Termination of sub-T Xpr2 The initial plasmid pYLXP' can be digested with the restriction endonucleases SnaBI and KpnI, followed by Gibson assembly technology to insert one or more fragments. The plasmid map of pYLXP' is shown below. Figure 1 As shown.

[0082] The initial plasmid pYLXP' was recovered by agarose gel electrophoresis after double digestion with the restriction endonucleases SnaBI and KpnI from TaKaRa.

[0083] Using the synthesized Gh2PS encoding gene as a template, and XP-Gh2PS-F and XP-Gh2PS-R as primers, the Gh2PS gene was amplified. The PCR amplification system consisted of 10 μL of 2×Phanta Max Master Mix (Dye Plus), 2 μL of template, 0.8 μL of upstream primer, 0.8 μL of downstream primer, and 6.4 μL of distilled water. The PCR amplification program was: denaturation at 95℃ for 15 s, annealing at 60℃ for 15 s, and extension at 72℃ (extension time = target fragment length / 1 kb, in min), repeated for 34 cycles.

[0084] use The Gel Extraction Kit (purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.) was used to purify and recover the Gh2PS gene fragment.

[0085] The linearized initial plasmid pYLXP' and the purified and recovered Gh2PS gene were assembled using the Seamless Cloning Kit from Shanghai Beyotime Biotechnology Co., Ltd. The Gh2PS gene was then inserted into the promoter P of plasmid pYLXP'. TEFin and Termination T Xpr2 Between these, the Gh2PS gene expression cassette (P) is assembled. TEFin -Gh2PS-T Xpr2 The Gibson-assembled reaction system consisted of: 7.5 μL of 2X Seamless Cloning Mix, x ng of linearized vector (pYLXP'), y ng of insert fragment (Gh2PS), and distilled water to a final volume of 15 μL. The amounts of linearized vector (x) and insert fragment (y) used could be calculated using the following formula: Optimal amount per fragment or linearized vector = [0.02 × number of base pairs of the fragment or linearized vector] ng. After incubating the reaction system at 50 °C for 30 min, a cyclic recombinant vector was obtained.

[0086] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, and the recombinant plasmid pYLXP'-Gh2PS was obtained by ampicillin-resistant plate screening and colony PCR and sequencing verification.

[0087] The recombinant plasmid pYL31-SfADC uses the initial plasmid pYL31 as a backbone, with the SfADC gene inserted into the promoter P of plasmid pYL31. FBA and Termination T Pex20 Between these, the SfADC gene expression cassette (P) is assembled. FBA -SfADC-T Pex20 The initial plasmid pYL31 contains the endogenous promoter P from Yarrowia lipolytica. FBA Termination of sub-T Pex20 It can be inserted into one or more fragments by digestion with the restriction endonuclease BamHI and then using Gibson assembly technology.

[0088] The recombinant plasmid pYL24-EcMAO uses the initial plasmid pYL24 as a backbone, with the EcMAO gene inserted into the promoter P of plasmid pYL24. TDH1 and Termination T Mig1 Between these, the EcMAO gene expression cassette (P) is assembled. TDH1 -EcMAO-T Mig1The initial plasmid pYL24 contains the endogenous promoter P from Yarrowia lipolytica. TDH1 Termination of sub-T Mig1 It can be inserted into one or more fragments by double digestion with restriction endonucleases XbaI and KpnI and then using Gibson assembly technology.

[0089] The recombinant plasmid pYLXP'-(CaMCR-C) uses the initial plasmid pYLXP' as a backbone, with the CaMCR-C gene inserted into the promoter P of plasmid pYLXP'. TEFin and Termination T Xpr2 Between these, the CaMCR-C gene expression cassette (P) is assembled. TEFin -CaMCR-CT Xpr2 ).

[0090] The recombinant plasmids pYL31-SfADC, pYL24-EcMAO, and pYLXP'-(CaMCR-C) were constructed according to the above-described method for constructing recombinant plasmid pYLXP'-Gh2PS.

[0091] 3. Construction of recombinant plasmid pUrLp-ΔDga1-(CaMCR-C)-SfADC-EcMAO

[0092] The recombinant plasmid pUrLp-ΔDga1-(CaMCR-C)-SfADC-EcMAO uses pUrLp as a backbone and inserts a 1kb homologous arm Dga1-up upstream of the start codon and a 1kb homologous arm Dga1-dn downstream of the stop codon at the Dga1 site (YALI0E32769g) in Yarrowia lipolytica Po1fk. A CaMCR-C gene expression cassette (P) is also inserted between these upstream and downstream homologous arms. TEFin -CaMCR-CT Xpr2 ), SfADC gene expression cassette (P FBA -SfADC-T Pex20 EcMAO gene expression cassette (P) TDH1 -EcMAO-T Mig1 Two loxP tags and the orotate nucleoside-5'-phosphate decarboxylase-encoding gene expression cassette (loxP-P) TEFin -URA3-T Xpr2 -loxP) is also present between the upstream and downstream homologous arms of the Dga1 site.

[0093] The initial plasmid pUrLp contains two loxP tags and an orotate nucleoside-5'-phosphate decarboxylase-encoding gene expression cassette (loxP-P). TEFin -URA3-T Xpr2Using the Gibson assembly technique described above in this embodiment, a 1kb homologous arm Dga1-up upstream of the start codon and a 1kb homologous arm Dga1-dn downstream of the stop codon at the Dga1 site can be sequentially inserted into the initial plasmid pUrLp, thereby obtaining the site-integrated plasmid pUrLp-ΔDga1. The plasmid map of pUrLp-ΔDga1 is shown below. Figure 2 As shown.

[0094] The site-integrating plasmid pUrLp-ΔDga1 was double-digested with restriction endonucleases NheI / SalI to recover the linearized site-integrating plasmid pUrLp-ΔDga1. Then, the plasmids carrying the target gene expression cassettes constructed based on pYLXP', pYL31, and pYL24 (recombinant plasmids pYL31-SfADC, pYL24-EcMAO, and pYLXP'-(CaMCR-C)) were double-digested with restriction endonucleases AvrII / SalI to recover the linearized target gene expression cassette. Since NheI and AvrII are isosigases, the linearized site-integrating plasmid pUrLp-ΔDga1 and the linearized target gene expression cassette can be ligated into a complete recombinant plasmid by T4 DNA ligase. Through multiple ligations, recombinant plasmids that simultaneously express multiple target genes can be constructed.

[0095] The specific construction steps are as follows:

[0096] (1) After digesting the plasmid pUrLp-ΔDga1 with restriction endonucleases NheI and SalI from TaKaRa, the linearized pUrLp-ΔDga1 plasmid was recovered by agarose gel electrophoresis.

[0097] The recombinant plasmid pYLXP'-(CaMCR-C) was digested with the restriction endonucleases AvrII and SalI from TaKaRa. The linearized CaMCR-C gene expression cassette (P) was recovered by agarose gel electrophoresis. TEFin -CaMCR-CT Xpr2 ).

[0098] Linearized pUrLp-ΔDga1 plasmid and CaMCR-C gene expression cassette (P TEFin -CaMCR-CT Xpr2 The recombinant vector was obtained by ligation using T4 DNA Ligase from Shanghai Beyotime Biotechnology Co., Ltd. The T4 DNA Ligase reaction system consisted of: 2 μL T4 DNA Ligase Mix, x ng linearized vector (pUrLp-ΔDga1), and insert fragment (P... TEFin -CaMCR-CT Xpr2Add distilled water to bring the volume to 10 μl. The amount of linearized vector (x) and insert fragment (y) used can be calculated using the following formula: Optimal amount of each fragment or linearized vector = [0.02 × number of base pairs of the fragment or linearized vector] ng.

[0099] The circular recombinant vector was transformed into E. coli DH5α competent cells. The positive recombinant plasmid pUrLp-ΔDga1-(CaMCR-C) was obtained by ampicillin-resistant plate screening, enzyme digestion verification, and sequencing verification.

[0100] (2) After digesting the plasmid pUrLp-ΔDga1-(CaMCR-C) with restriction endonucleases NheI and SalI from TaKaRa, the linearized pUrLp-ΔDga1-(CaMCR-C) plasmid was recovered by agarose gel electrophoresis.

[0101] After digesting plasmid pYL31-SfADC with restriction endonucleases AvrII and SalI from TaKaRa, the linearized SfADC gene expression cassette (P) was recovered by agarose gel electrophoresis. FBA -SfADC-T Pex20 ).

[0102] The linearized pUrLp-ΔDga1-(CaMCR-C) plasmid and SfADC gene expression cassette (P FBA -SfADC-T Pex20 The T4 DNA Ligase from Shanghai Beyotime Biotechnology Co., Ltd. was used to achieve ligation, resulting in a circular recombinant vector. The T4 DNA Ligase reaction system is as described above.

[0103] The circular recombinant vector was transformed into E. coli DH5α competent cells. The positive recombinant plasmid pUrLp-ΔDga1-(CaMCR-C)-SfADC was obtained by ampicillin-resistant plate screening, enzyme digestion verification and sequencing verification.

[0104] (3) The plasmid pUrLp-ΔDga1-(CaMCR-C)-SfADC was digested with restriction endonucleases NheI and SalI from TaKaRa, and the linearized pUrLp-ΔDga1-(CaMCR-C)-SfADC plasmid was recovered by agarose gel electrophoresis.

[0105] After digesting plasmid pYL24-EcMAO with the restriction endonucleases AvrII and SalI from TaKaRa, the linearized EcMAO gene expression cassette (P) was recovered by agarose gel electrophoresis. TDH1 -EcMAO-T Mig1).

[0106] The linearized pUrLp-ΔDga1-(CaMCR-C)-SfADC plasmid and the EcMAO gene expression cassette (P) were used to express the linearized pUrLp-ΔDga1-(CaMCR-C)-SfADC plasmid and the EcMAO gene expression cassette (P TDH1 -EcMAO-T Mig1 The T4 DNA Ligase from Shanghai Beyotime Biotechnology Co., Ltd. was used to achieve ligation, resulting in a circular recombinant vector. The T4 DNA Ligase reaction system is as described above.

[0107] The circular recombinant vector was transformed into *E. coli* DH5α competent cells. After ampicillin-resistant plate selection and verification via enzyme digestion and sequencing, the positive recombinant plasmid pUrLp-ΔDga1-(CaMCR-C)-SfADC-EcMAO was obtained. The plasmid map of recombinant plasmid pUrLp-ΔDga1-(CaMCR-C)-SfADC-EcMAO is shown below. Figure 3 As shown.

[0108] The plasmid pUrLp-ΔDga1-(CaMCR-C)-SfADC-EcMAO was digested with the restriction endonuclease AvrII from TaKaRa, and the linearized pUrLp-ΔDga1-(CaMCR-C)-SfADC-EcMAO plasmid was recovered by agarose gel electrophoresis. This linearized plasmid can be used for the subsequent construction of recombinant Yersinia lipolytica strains.

[0109] 4. Construction of recombinant plasmid pUrLp-ΔLip1-Gh2PS

[0110] The recombinant plasmid pUrLp-ΔLip1-Gh2PS uses pUrLp as a backbone and inserts a 1kb homologous arm upstream of the start codon (Lip1-up) and a 1kb homologous arm downstream of the stop codon (Lip1-dn) from the Lip1 site in Yarrowia lipolytica Po1fk. A Gh2PS gene expression cassette (P) is also inserted between these upstream and downstream homologous arms. TEFin -Gh2PS-T Xpr2 Two loxP tags and the orotate nucleoside-5'-phosphate decarboxylase-encoding gene expression cassette (loxP-P) TEFin -URA3-T Xpr2 -loxP) is also present between the upstream and downstream homologous arms of the Lip1 site.

[0111] The recombinant plasmid pUrLp-ΔDga1-(CaMCR-C)-SfADC-EcMAO was constructed using the same method. The plasmid map of pUrLp-ΔDga1-(CaMCR-C)-SfADC-EcMAO is shown below. Figure 4 As shown.

[0112] The plasmid pUrLp-ΔLip1-Gh2PS was digested with the restriction endonuclease AvrII from TaKaRa, and the linearized pUrLp-ΔLip1-Gh2PS plasmid was recovered by agarose gel electrophoresis. This linearized plasmid can be used for the subsequent construction of recombinant Yersinia lipolytica strains.

[0113] 5. Construction of recombinant plasmid pUrLp-ΔDga1-SfADC

[0114] (1) After digesting the plasmid pUrLp-ΔDga1 with restriction endonucleases NheI and SalI from TaKaRa, the linearized pUrLp-ΔDga1 plasmid was recovered by agarose gel electrophoresis.

[0115] After digesting plasmid pYL31-SfADC with restriction endonucleases AvrII and SalI from TaKaRa, the linearized SfADC gene expression cassette (P) was recovered by agarose gel electrophoresis. FBA -SfADC-T Pex20 ).

[0116] Linearized pUrLp-ΔDga1 plasmid and SfADC gene expression cassette (P FBA -SfADC-T Pex20 The recombinant vector was obtained by ligation using T4 DNA Ligase from Shanghai Beyotime Biotechnology Co., Ltd. The T4 DNA Ligase reaction system consisted of: 2 μL T4 DNA Ligase Mix, x ng linearized vector (pUrLp-ΔDga1), and insert fragment (P... FBA -SfADC-T Pex20 Add distilled water to bring the volume to 10 μl. The amount of linearized vector (x) and insert fragment (y) used can be calculated using the following formula: Optimal amount of each fragment or linearized vector = [0.02 × number of base pairs of the fragment or linearized vector] ng.

[0117] The circular recombinant vector was transformed into *E. coli* DH5α competent cells. Positive recombinant plasmids pUrLp-ΔDga1-SfADC were obtained through ampicillin-resistant plate selection, enzyme digestion verification, and sequencing verification. The plasmid map of recombinant plasmid pUrLp-ΔDga1-SfADC is shown below. Figure 5 As shown.

[0118] The plasmid pUrLp-ΔDga1-SfADC was digested with the restriction endonuclease AvrII from TaKaRa, and the linearized pUrLp-ΔDga1-SfADC plasmid was recovered by agarose gel electrophoresis. This linearized plasmid can be used for the subsequent construction of recombinant Yersinia lipolytica strains.

[0119] Example 2

[0120] Construction of recombinant Yersinia lipophila strain.

[0121] 1. Construction of recombinant strain 1

[0122] The linearized plasmid pUrLp-ΔLip1-Gh2PS containing the Gh2PS gene expression cassette prepared in Example 1 was introduced into Yarrowia lipolytica Po1fk. The Gh2PS gene expression cassette was integrated into the Lip1 site of the genome via homologous recombination, resulting in a positive clone. Then, a loxP tag and the orotate nucleoside-5'-phosphate decarboxylase-encoded gene expression cassette (P) were lost using Cre-loxP technology. TEFin -URA3-T Xpr2 After that, recombinant bacteria 1 was obtained. The specific method is as follows:

[0123] (1) Yarrowia lipolytica Po1fk was cultured overnight in YPD liquid medium (water, 2% peptone, 1% yeast extract and 2% glucose). After centrifuging 1 mL of the overnight culture at low speed, the supernatant was discarded. The cell pellet, 95 μL of 50% PEG4000 and 5 μL of 2M lithium acetate solution were thoroughly mixed to form competent cells.

[0124] (2) After thoroughly mixing 5 μL of the recovered linearized plasmid pUrLp-ΔLip1-Gh2PS and 5 μL of boiled denatured fish sperm DNA (ssDNA), the mixture was added to the competent cells prepared in the previous step. The cells were incubated at 30°C for 45 minutes, with vortexing for 15 seconds every 15 minutes. Then, the cells were heat-shocked at 39°C for 10 minutes. The heat-shocked competent cells were plated onto appropriate selection plates. Positive clones were screened using CSM-Ura selection medium and identified by colony PCR. Positive clones that passed PCR were inoculated into YPD liquid medium and cultured overnight to prepare competent cells. The circular plasmid pYLXP'-Cre was introduced into the competent cells of the positive clones. The Cre enzyme expressed in plasmid form could recognize two loxP tags with identical sequences on the genome, causing homologous recombination between the two loxP tags, resulting in the loss of one loxP tag and the orotate nucleoside-5'-phosphate decarboxylase-encoding gene expression cassette (P...). TEFin -URA3-T Xpr2This process restores the Ura nutrient selection marker. Competent cells were then plated on CSM-Leu selection medium and incubated at 30°C for 2 days. Single colonies from the CSM-Leu plates were simultaneously streaked onto YPD plates containing 5-fluoroorotic acid and CSM-Ura plates to observe cell growth. While 5-fluoroorotic acid itself is non-toxic to yeast cells, the functional Ura3 gene encodes orotate nucleoside-5'-monophosphate decarboxylase (OMP decarboxylase), which converts 5-fluoroorotic acid into a toxic form (such as 5-fluorouracil), thus preventing yeast cell growth. Therefore, after the Ura3 gene is lost in step 5, cells without the Ura3 gene can survive. Single colonies that grew on YPD plates containing 5-fluoroorotic acid but not on CSM-Ura plates were selected for colony PCR verification. Colonies that were successfully validated by PCR were inoculated into YPD liquid medium. After overnight incubation, the circular plasmid pYLXP'-Cre was lost, thus restoring the Leu nutrient selection marker. The strain that simultaneously restored both the Ura and Leu nutrient selection markers was named recombinant strain 1.

[0125] 2. Construction of recombinant bacteria 2

[0126] The linearized plasmid pUrLp-ΔDga1-(CaMCR-C)-SfADC-EcMAO containing the CaMCR-C, SfADC, and EcMAO gene expression cassettes was introduced into recombinant bacteria 1. The CaMCR-C, SfADC, and EcMAO gene expression cassettes were then integrated into the Dga1 site of the genome via homologous recombination, resulting in recombinant bacteria 2. The construction steps were the same as for recombinant bacteria 1.

[0127] 3. Construction of recombinant strain 3

[0128] The linearized recombinant plasmid pUrLp-ΔDga1-SfADC containing the SfADC gene expression cassette was introduced into recombinant bacteria 1. The SfADC gene expression cassette was then integrated into the Dga1 site of the genome via homologous recombination to construct recombinant bacteria 3. The construction steps were the same as those for recombinant bacteria 2.

[0129] Example 3

[0130] Preparation of 4-hydroxy-6-hydroxyethyl-2-pyranone (HHEP). HHEP was produced by fermentation using a recombinant Yersinia lipolyticis strain. Fermentation was carried out using recombinant strain 2 from Example 2.

[0131] 1. Culture of recombinant bacteria

[0132] The activated recombinant strain 2 was cultured in YPD liquid medium (water, 2% peptone, 1% yeast extract, 2% glucose) at 30℃ and 250 rpm for 16 h to obtain seed culture. The seed culture was inoculated into 25 mL of fermentation medium to achieve an initial OD600 of 0.2, and cultured at 30℃ and 250 rpm with shaking for 5 days to obtain fermentation broth. The fermentation medium (YPD80 medium) formulation was: water, glucose 80 g / L, yeast extract 10 g / L, and peptone 20 g / L.

[0133] 2. Treatment of fermentation broth

[0134] 100 μL of fermentation broth was resuspended in 700 μL of aqueous solution containing the grinding particles. The mixture was then ground using a cryogenic grinder at -20°C for 20 cycles (40 seconds per cycle), followed by centrifugation at 12,000 rpm for 3 minutes. The supernatant was then analyzed by LCMS and HPLC.

[0135] 3. LCMS Analysis

[0136] Qualitative analysis of the supernatant was performed using liquid chromatography-mass spectrometry (LC-MS), and characterization was performed using a Thermo Scientific QExtractive mass spectrometer equipped with an electrospray ionization (ESI) source, operating in both positive and negative ion modes. Full-scan mass spectra were acquired from a mass-to-charge ratio (m / z) of 50 to 500. The optimized ESI parameters were as follows: spray voltage 3500 V, capillary temperature 350 °C, sheath gas flow rate 30 Arb, auxiliary gas flow rate 10 Arb, and probe heating temperature 325 °C. Chromatographic separation was performed using a C18 column (2.1 mm × 100 mm), a flow rate of 0.5 mL / min, and an injection volume of 5 μL. The mobile phase consisted of phase A (an aqueous solution containing 0.1% formic acid) and phase B (methanol), with a gradient elution program as follows: initial phase B ratio of 10%, linearly increased to 100% within 20 minutes, maintained for 3 minutes, and then returned to the initial ratio of 10% at 25 minutes. The flow rate was set to 1.0 mL / min, the column temperature to 30 °C, the detection wavelength to 280 nm, and the injection volume to 10 μL.

[0137] 4. High-performance liquid chromatography (HPLC) analysis

[0138] An Agilent 1260 Infinity II high-performance liquid chromatography (HPLC) system equipped with a variable wavelength detector (VWD) and a C18 column (4.6 mm × 100 mm) was used. The mobile phase consisted of phase A (an aqueous solution containing 0.1% formic acid) and phase B (methanol), with the following gradient elution program: the initial proportion of phase B was 10%, linearly increased to 100% within 20 minutes, maintained for 3 minutes, and then returned to the initial proportion of 10% at 25 minutes. The flow rate was set at 1.0 mL / min, the column temperature at 30 °C, the detection wavelength at 280 nm, and the injection volume at 10 μL. Triacetic acid lactone (TAL), provided by Shanghai Maclean Biochemical Technology Co., Ltd., was used as an internal standard for the quantitative analysis of HHEP. The retention times of HHEP and TAL were approximately 1.2 min and 4.8 min, respectively.

[0139] The HPLC chromatogram of the supernatant is as follows: Figure 6 As shown, the results indicate that a main peak appears at a retention time of 1.23 min, and a peak of triacetic acid lactone (TAL) appears at a retention time of 4.83 min. Based on the synthetic pathway of triacetic acid lactone (TAL), the structure of the main peak substance is deduced, and the deduced structural formula of the main peak substance is shown in formula (I).

[0140]

[0141] The main peak substance was named 4-hydroxy-6-hydroxyethyl-2-pyrone (HHEP) using systematic nomenclature.

[0142] A comparison of the synthesis of TAL and HHEP is shown in the figure below. Figure 7 As shown. The LCMS spectrum of TAL is as follows. Figure 8 As shown.

[0143] After mass spectrometry analysis of the main peak, the main peak mass spectrum (LCMS spectrum of HHEP) is as follows: Figure 9 As shown, a major peak with a mass-to-charge ratio (m / z) of 157.07 was obtained, which is highly consistent with the theoretical m / z value of 157.05 for the predicted substance HHEP, indicating that the product HHEP has been successfully generated. In addition, several characteristic fragment peaks were detected, such as m / z = 72.14, 83.12, 84.12, 102.19, 112.09, and 124.07, which are characteristic fragment ions of HHEP, further confirming the formation of HHEP.

[0144] HHEP mass spectrometry fragment information such as Figure 10 As shown, the results are consistent with Figure 9 The characteristic fragment ions of HHEP are consistent.

[0145] The de novo synthesis pathway of HHEP is as follows: Figure 11 As shown.

[0146] The synthesis of HHEP in this invention reconstructs the malonyl-CoA and 3-hydroxypropionyl-CoA biosynthetic pathways derived from aspartate metabolism in the cellular metabolism of *Yarrowia lipolytica* strain, and integrates a heterologous polyketide synthase (2-pyranone synthase) functional module. The malonyl-CoA pathway is achieved through the cascade expression of aspartate decarboxylase (SfADC), β-alanine pyruvate aminotransferase (YlBAPAT), monoamine oxidase (EcMAO), and the C-terminus of malonyl-CoA reductase (…). CaMCR-C directs the aspartate metabolic flux to malonyl-CoA. The 3-hydroxypropionyl-CoA pathway utilizes the synergistic action of 3-hydroxypropionyl-CoA dehydrogenase (YlHBD1) and 3-hydroxyisobutyryl-CoA hydrolase (YlEHD3) to direct the malonate hemialdehyde metabolic flux to 3-hydroxypropionyl-CoA. The heteropolyketide synthase (2-pyranone synthase) functional module catalyzes the condensation cyclization of 3-hydroxypropionyl-CoA and malonyl-CoA to generate HHEP.

[0147] Example 4

[0148] Performance testing of HHEP production by the strain. The initial strain Yarrowia lipolytica Po1fk (initial strain Po1fk), recombinant strains 1, 2, and 3 from Example 2 were used as experimental groups 1-4, respectively. Recombinant strain 2 was used with an additional 34 mM aspartic acid added to the fermentation medium as experimental group 5. The performance of the strain in producing HHEP was tested.

[0149] 1. Culture of recombinant bacteria

[0150] Strains from experimental groups 1-5 were activated and cultured in YPD liquid medium (water, 2% peptone, 1% yeast extract, 2% glucose) at 30℃ and 250 rpm for 16 h to obtain seed culture. The seed culture was inoculated into 25 mL of fermentation medium to achieve an initial OD600 of 0.2, and cultured at 30℃ and 250 rpm with shaking for 5 days to obtain the fermentation broth. The fermentation medium (YPD80 medium) formulation was: water, glucose 80 g / L, yeast extract 10 g / L, and peptone 20 g / L.

[0151] 2. Analysis of fermentation broth

[0152] 100 μL of fermentation broth was resuspended in 700 μL of aqueous solution containing the grinding particles. The mixture was then ground using a cryogenic grinder at -20°C for 20 cycles (40 seconds per cycle), followed by centrifugation at 12,000 rpm for 3 minutes. The supernatant was then analyzed by high-performance liquid chromatography (HPLC).

[0153] The LCMS and HPLC analysis methods are the same as in Example 3.

[0154] Qualitative analysis results of HHEP production by recombinant bacterial fermentation are as follows: Figure 12 As shown, the results indicated that recombinant strain 2 exhibited a significant peak of the target product HHEP at a retention time of approximately 1.2 min, while this peak was not detected in the initial strain Po1fk or recombinant strain 1, suggesting that HHEP synthesis depends on the construction of a specific metabolic module. Furthermore, recombinant strains 1 and 2 showed a significant peak of the byproduct triacetin (TAL) at a retention time of approximately 4.83 min. This is because 2-pyranone synthase (Gh2PS) exhibits multiple reactivity, catalyzing both HHEP and TAL formation. After the addition of 34 mM aspartic acid, the peak areas of both HHEP and TAL in recombinant strain 2 increased, indicating that the addition of aspartic acid can improve the HHEP yield of recombinant strain 2.

[0155] Quantitative analysis results of HHEP production by recombinant bacterial fermentation are as follows: Figure 13 As shown, the results indicated that, using glucose as the sole carbon source, after 5 days of fermentation in a 250 mL shake flask, the recombinant strain 2 produced a final yield of 3.1 g / L of 4-hydroxy-6-hydroxyethyl-2-pyranone (HHEP) and a final yield of 1.2 g / L of the byproduct triacetin (TAL), meaning a yield of 3.1 g of HHEP per liter of fermentation broth. This demonstrated the efficient synthesis of the novel pyranone derivative HHEP in *Yarrowia lipolytica*. Adding 34 mM aspartic acid to the culture medium further increased the HHEP yield of recombinant strain 2 to 3.6 g / L and the TAL yield to 1.4 g / L, indicating that the addition of aspartic acid can improve the HHEP yield of recombinant strain 2.

[0156] The specific results of HHEP and TAL production of recombinant strains 2 and 3 are shown in Table 3 below.

[0157] Table 3

[0158]

[0159] The results show that the recombinant strain 2 constructed in Example 2 can express aspartate decarboxylase (SfADC), monoamine oxidase (EcMAO), malonyl-CoA reductase C-terminus (CaMCR-C), and 2-pyranone synthase (Gh2PS). Recombinant strain 2 exhibits a high HHEP yield after fermentation, reaching 3.1 g / L. The HHEP yield obtained by fermentation with the additional addition of 34 mM aspartate is significantly higher than that of the fermentation group without aspartate. Based on recombinant strain 1, expressing only SfADC is sufficient to achieve HHEP synthesis in *Yarrowia lipolytica*.

[0160] This method achieves the first efficient microbial synthesis of HHEP, providing a disruptive technology platform for the industrial production of novel pyranone derivatives.

[0161] The inventors further experimented with the fermentation culture formula, using recombinant strain 2 for fermentation. The results showed that the fermentation culture medium formula of water, glucose 20-80 g / L, yeast extract 1-10 g / L, and peptone 2-20 g / L could all produce HHEP.

[0162] The fermentation medium formula is: water, glucose 20-80 g / L, yeast extract 1-10 g / L, peptone 2-20 g / L, aspartic acid 10-1000 mM, which can also ferment and produce HHEP.

[0163] Example 5

[0164] Antibacterial activity test of fermentation broth. The initial strain Yarrowia lipolytica Po1fk (initial strain Po1fk), recombinant strain 1 and recombinant strain 2 from Example 2 were used as experimental groups ac, and the antibacterial activity of the fermentation broth of the strains was tested.

[0165] The strain ac from the activation experimental group was cultured in YPD liquid medium (water, 2% peptone, 1% yeast extract, 2% glucose) at 30℃ and 250 rpm for 16 h to obtain seed culture. The seed culture was inoculated into 25 mL of fermentation medium to achieve an initial OD600 of 0.2, and cultured at 30℃ and 250 rpm with shaking for 5 days to obtain the fermentation broth. The OD600 of the fermentation broth was measured at different time points, and growth curves of the strain were plotted.

[0166] Fermentation media include YPD20 medium and YPD80 medium. The formula for YPD20 medium is: water, glucose 20 g / L, yeast extract 10 g / L, and peptone 20 g / L. The formula for YPD80 medium is: water, glucose 80 g / L, yeast extract 10 g / L, and peptone 20 g / L.

[0167] Comparison of growth curves of the strain in different culture media: Figure 14 As shown, the results indicate that, in both YPD20 and YPD80 media, the growth of recombinant strain 2 was significantly inhibited compared to the initial strain Po1fk and recombinant strain 1. This suggests that the fermentation product of recombinant strain 2 has an inhibitory effect on the growth of this strain, and also indicates that HHEP produced by recombinant strain 2 has a significant inhibitory effect on the growth of the strain, suggesting that HHEP may be a potential antibacterial agent.

[0168] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A pyranone derivative, characterized in that, Its name is 4-hydroxy-6-hydroxyethyl-2-pyranone, and its structural formula is shown in formula (I). 2.2 Application of the pyranone synthase encoding gene and the aspartate decarboxylase encoding gene in the preparation of recombinant engineered bacteria for producing the pyranone derivatives of claim 1; Alternatively, the application of the 2-pyranone synthase encoding gene, along with the aspartate decarboxylase encoding gene, the monoamine oxidase encoding gene, and the malonyl-CoA reductase encoding gene, in the preparation of recombinant engineered bacteria that produce the pyranone derivative of claim 1. Its features are, The nucleotide sequence of the 2-pyranone synthase encoding gene is shown in SEQ ID NO:4; the nucleotide sequence of the aspartate decarboxylase encoding gene is shown in SEQ ID NO:1; the nucleotide sequence of the monoamine oxidase encoding gene is shown in SEQ ID NO:2; and the nucleotide sequence of the malonyl-CoA reductase encoding gene is shown in SEQ ID NO:

3.

3. A recombinant expression vector, characterized in that, The recombinant expression vector includes a 2-pyranone synthase gene expression cassette and an aspartate decarboxylase gene expression cassette. The 2-pyranone synthase gene expression cassette includes a promoter, a Gh2PS encoding gene, and a terminator; the nucleotide sequence of the Gh2PS encoding gene is shown in SEQ ID NO:4; The aspartate decarboxylase gene expression cassette includes a promoter, an SfADC encoding gene, and a terminator; the nucleotide sequence of the SfADC encoding gene is shown in SEQ ID NO:

1.

4. The recombinant expression vector as described in claim 3, characterized in that, The recombinant expression vector also includes a monoamine oxidase gene expression cassette and a malonyl-CoA reductase gene expression cassette. The monoamine oxidase gene expression cassette includes a promoter, an EcMAO encoding gene, and a terminator, and the nucleotide sequence of the EcMAO encoding gene is shown in SEQ ID No:2; The malonyl-CoA reductase gene expression cassette includes a promoter, a CaMCR-C encoding gene, and a terminator; the nucleotide sequence of the CaMCR-C encoding gene is shown in SEQ ID No:3; And / or, the promoter is P TEFin P FBA or P TDH1 Any one of them; And / or, the terminator is T Xpr2 T Pex20 or T Mig1 Any one of them.

5. A recombinant engineered bacterium, characterized in that, The recombinant expression vector described in claim 3 or 4 is transferred into a host bacterium to construct the recombinant engineered bacterium.

6. A method for producing the pyranone derivative of claim 1, characterized in that, The recombinant engineered bacteria described in claim 5 were inoculated into a fermentation medium for fermentation culture to obtain a fermentation product containing 4-hydroxy-6-hydroxyethyl-2-pyranone; The fermentation medium is formulated as follows: water, glucose 20-80 g / L, yeast extract 1-10 g / L and peptone 2-20 g / L; Preferably, the fermentation medium is formulated as follows: water, glucose 20-80 g / L, yeast extract 1-10 g / L, peptone 2-20 g / L and aspartic acid 10-1000 mM.

7. The fermentation product prepared by the method of claim 6.

8. The use of the fermentation product of claim 7 as an antibacterial agent.

9. The use of the pyranone derivative of claim 1 as an antibacterial agent.

10. An antibacterial agent, characterized in that, Includes the pyranone derivative of claim 1 or the fermentation product of claim 7.

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