Method for producing vanillin by using natural lignocellulose biomass through pichia pastoris co-culture

Abstract

The invention relates to a method for producing vanillin from natural lignocellulose biomass through co-culture of pichia pastoris, which comprises the following steps: respectively constructing a xylan hydrolysis recombinant strain (for releasing ferulic acid) and a vanillin synthesis recombinant strain (for converting ferulic acid) to realize conversion production from a natural xylan substrate to vanillin. In order to reduce the generation of by-products, 15 genes for coding aldehyde dehydrogenase (ALDRs), alcohol dehydrogenase (ADHs) and aldehyde ketoreductase (AKRs) are further knocked out from the vanillin synthesis recombinant strain, so that the vanillin can be further converted into the by-product vanillic acid, thereby improving the yield and purity of the vanillin.

Description

Technical Field

[0001] The present invention belongs to the field of microbial engineering, and specifically relates to a method for producing vanillin by co-culturing Pichia pastoris and utilizing natural lignocellulose biomass. Background Art

[0002] Vanillin is one of the world's most produced broad-spectrum flavorings, widely used in food, medicine, daily chemicals, and agriculture. Compared to traditional plant extraction and mainstream chemical synthesis methods, biosynthesis of vanillin using microbial cell factories, which utilize renewable natural substrates as raw materials, offers near-natural quality and lower production costs. This approach aligns with the development of green environmental protection and meets downstream market demand for safe, "natural" products, resulting in continued global demand.

[0003] Xylan, a hemicellulose component abundant in plant cell walls, is rich in carbohydrates and aromatic polymers and is considered an ideal renewable feedstock for the production of biofuels and other high-value compounds. Xylan can be hydrolyzed into functional prebiotics such as glucose, xylose, or xylo-oligosaccharides and manno-oligosaccharides under the synergistic catalysis of multiple enzymes. It can also be further converted into high-value fine chemicals. Furthermore, xylan contains numerous phenolic acid substituents, which can be released through targeted hydrolysis to produce products such as ferulic acid. These products, in addition to their inherent high economic value, can also serve as intermediates for the synthesis of other high-value compounds such as vanillin, catechol, and curcumin. Therefore, developing food-grade cell factories using xylan as a substrate to produce vanillin is an important approach for the high-value utilization of agricultural product processing byproducts and the green and efficient production of vanillin. However, traditional single-bacterial fermentation methods suffer from complex metabolic pathways and low product yields, making the efficient conversion of xylan to vanillin difficult.

[0004] In recent years, microbial co-culture technology has demonstrated tremendous potential in biosynthesis. By constructing functionally complementary strains, co-culture systems can achieve the division of labor and collaboration in complex metabolic pathways, thereby increasing the yield and conversion efficiency of target products. However, food-grade microbial co-culture technology for the conversion of xylan to vanillin remains unavailable. Therefore, the development of an efficient and stable food-grade "xylan hydrolysis-vanillin synthesis" co-culture system has significant scientific and practical value. Summary of the Invention

[0005] The present invention provides a co-cultivation method, wherein a recombinant strain capable of hydrolyzing xylan to release ferulic acid and a recombinant strain capable of utilizing ferulic acid to produce vanillin are respectively constructed. The co-cultivation of the two strains realizes the synthesis of vanillin using xylan as a substrate.

[0006] The first aspect of the present invention is to provide a recombinant Pichia pastoris that can be used for the synthesis of vanillin.

[0007] Heterologous expression of the key vanillin biosynthesis enzyme genes FCS and ECH in wild-type Pichia pastoris can convert ferulic acid into vanillin, but testing has shown that the synthesized vanillin will be further degraded into vanillic acid and vanillyl alcohol within 12 hours.

[0008] Therefore, the inventors made an improvement by knocking out one or more of the endogenous genes for aldehyde dehydrogenases (ALDRs), alcohol dehydrogenases (ADHs), and aldehyde-keto reductases (AKRs) in the recombinant Pichia pastoris. This recombinant manipulation aims to block the conversion of vanillin to vanillic acid and vanillyl alcohol in the recombinant Pichia pastoris, thereby reducing the degradation loss of vanillin.

[0009] Furthermore, the Pichia pastoris simultaneously knocks out genes for aldehyde dehydrogenases (ALDRs), alcohol dehydrogenases (ADHs) and aldehyde-keto reductases (AKRs).

[0010] Furthermore, the knocked-out aldehyde dehydrogenase (ALDRs) encoding genes include at least one of PAS_chr3_0987, PAS_chr2-1_0453, PAS_chr4_0043, PAS_chr4_0470, and PAS_chr2-1_0853;

[0011] Furthermore, the knocked-out alcohol dehydrogenase (ADHs) encoding gene includes at least one of PAS_chr3_0006, PAS_chr4_0576, PAS_chr1-1_0357, PAS_chr2-1_0313, PAS_chr2-1_0472, and PAS_chr3_1028;

[0012] Furthermore, the knocked-out aldehyde-keto reductase (AKRs) encoding genes include at least one of PAS_chr1-1_0360, PAS_chr2-1_0573, PAS_chr4_0336, and PAS_chr3_0446.

[0013] Specifically, the gene sequence is shown in the following table

[0014] Table 1

[0015]

[0016]

[0017] Furthermore, Pichia pastoris is recombined to express feruloyl-CoA synthase FCS protein and / or enoyl-CoA hydratase / aldolase ECH by genetic engineering methods.

[0018] The nucleotide sequence of the feruloyl-CoA synthase FCS protein is SEQ ID NO: 16, and the amino acid sequence is SEQ ID NO: 23;

[0019] The nucleotide sequence of the enoyl-CoA hydratase aldolase ECH is SEQ ID NO: 17, and the amino acid sequence is SEQ ID NO: 24;

[0020] The feruloyl-CoA synthase FCS and the enoyl-CoA hydratase / aldolase ECH are both derived from Amycolatopsis sp. ATCC 39116.

[0021] Furthermore, in order to improve the substrate affinity and catalytic efficiency of ECH, optionally, the 37th position of ECH can be modified with R37M. The modified nucleotide sequence is SEQ ID NO: 18, and the amino acid sequence is SEQ ID NO: 21, wherein R37 is the key site for the interaction between ECH and the substrate acetyl-CoA (forming an H bond). This optimization significantly improves the substrate affinity and catalytic efficiency of ECH through precise amino acid replacement. The modified ECH is expressed as "ECH R37M ", M in the sequence is the site after mutation, and the 37th position is counted from the second "S" of SEQ ID NO: 21, and the count does not include the M at the N-terminus of the sequence.

[0022] All genes were codon-optimized for Pichia pastoris; the recombinant Pichia pastoris was based on GS115 as the starting strain.

[0023] In a specific embodiment of the present invention, the recombinant Pichia pastoris is FCS (SEQ ID NO: 16), ECH / ECH R37M (SEQ ID NO: 17 or 18) is integrated into the genome of the starting strain GS115; specifically, it is integrated into the His site of the genome of the starting strain GS115. The construction method of the FCS and ECH is not limited. Each can be equipped with a promoter and terminator in the genome for independent expression, or can be constructed as a fusion protein such as FCH-ECH, ECH-FCH, or FCS-ECH. R37M , or ECH R37M -FCH, can be connected with or without a flexible linker to express the fusion protein through an expression vector.

[0024] Specifically, you can choose P AOX1 、P GAP 、P FLD1 、P ALD6 、P ADH2 、P PDC1 、P TEF1 As a promoter.

[0025] In a specific embodiment, the present invention provides a genetically engineered strain of Pichia pastoris EF, which is derived from GS115 as a starting strain and comprises ECH R37M The nucleotide sequences of SEQ ID NO: 1-15 are integrated into the His site of the genome of the starting strain GS115, and the genes of aldehyde dehydrogenase (ALDRs), alcohol dehydrogenase (ADHs) and aldehyde-keto reductase (AKRs) are further knocked out. The strain has the ability to efficiently synthesize vanillin. The second aspect of the present invention is to provide a recombinant Pichia pastoris that can be used for the hydrolysis of xylan in natural lignocellulosic biomass.

[0026] Recombinant Pichia pastoris overexpressed the bifunctional enzyme xylanase / feruloyl esterase rXyn10A / Fae1A.

[0027] The nucleotide sequence of the bifunctional enzyme xylanase / feruloyl esterase rXyn10A / Fae1A was codon-optimized in Pichia pastoris based on preference.

[0028] The bifunctional enzyme xylanase / feruloyl esterase rXyn10A / Fae1A is derived from the bacterial flora EMSD5.

[0029] Furthermore, the recombinant Pichia pastoris is based on GS115 as the starting strain.

[0030] Furthermore, the recombinant Pichia pastoris is obtained by integrating rXyn10A / Fae1A into the genome of the starting strain GS115; further, the rXyn10A / Fae1A is integrated into the AOX1 site of the genome of the starting strain GS115.

[0031] In a specific embodiment, the nucleotide sequence of xylanase / feruloyl esterase rXyn10A / Fae1A is shown in SEQ ID NO: 20, and the amino acid sequence is shown in SEQ ID NO: 22.

[0032] In a specific embodiment of the present invention, a Pichia pastoris XF is provided, wherein the Pichia pastoris is a recombinant strain capable of expressing the rXyn10A / Fae1A amino acid sequence; further, the Pichia pastoris is obtained by introducing a recombinant strain comprising the nucleotide sequence of SEQ ID NO: 20 comprising the bifunctional xylanase / feruloyl esterase rXyn10A / Fae1A into Pichia pastoris.

[0033] The aforementioned use of the recombinant Pichia pastoris in hydrolyzing natural cellulosic biomass can further efficiently hydrolyze natural lignocellulosic biomass including, but not limited to, one or more of corn cobs, corn stalks, rice straw, wheat straw, and wheat bran.

[0034] Furthermore, lignocellulosic biomass can be treated by pretreatment methods such as steam explosion, ultrafine grinding, ultrasonication, and mechanical grinding to improve the enzymatic accessibility of the side chain ferulic acid groups. Lignocellulosic biomass can also be treated by acid hydrolysis, enzymatic hydrolysis, alkaline hydrolysis, organic solvents, and microbial degradation.

[0035] The natural xylan substrate concentration is 2%-10%, preferably 2%, 4%, 6%, 8% and 10%, and can also be 20g / L-100g / L, preferably 20g / L, 40g / L, 60g / L, 80g / L and 100g / L.

[0036] The third object of the invention is to provide a co-cultivation system for producing vanillin by co-cultivating microorganisms using natural lignocellulosic biomass.

[0037] A co-culture system comprising a recombinant microorganism capable of hydrolyzing xylan to release ferulic acid and a recombinant microorganism capable of converting ferulic acid to synthesize vanillin, see Figure 8 ;

[0038] Furthermore, the recombinant microorganism can be selected from Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Bacillus subtilis, etc.; preferably, it can be Pichia pastoris.

[0039] Preferably, the recombinant Pichia pastoris involved in the first and second aspects of the present invention can be further selected;

[0040] Furthermore, genetically engineered Pichia pastoris XF and genetically engineered Pichia pastoris EF can be selected.

[0041] The seed liquid of xylan hydrolyzing recombinant Pichia pastoris and vanillin synthesizing Pichia pastoris is mixed in a ratio of (1-50):1, and then inoculated into a Pichia pastoris culture medium for fermentation to obtain a co-culture system; preferably, the inoculation ratio can be 1:1, 2:1, 3:1, 5:1, 10:1, 20:1, 30:1, or 50:1.

[0042] Carbon source: The carbon source used in the culture medium used in the co-cultivation system is at least one of methanol, glycerol, glucose, and sorbitol, preferably two of them can be selected; more specifically, when two are selected as carbon sources, one of the combinations of methanol-glycerol, methanol-glucose, and methanol-sorbitol can be selected, and the ratio of the two carbon sources is one of 1:1, 1:2, 2:1, 1:3, 3:1, 1:4, and 4:1.

[0043] Inoculation ratio: In one embodiment of the present invention, the fermentation conditions are as follows: the seed solution of the xylan hydrolysis module strain XF and the seed solution of the vanillin synthesis module strain EF are inoculated in the optimal culture medium methanol-glycerol at a ratio of 1:1 and fermented for 48-72 hours; wherein, pretreated natural lignocellulosic biomass is added 12-16 hours after fermentation, and methanol is added at a final concentration of 0.5%-1.5% every 24 hours for induction.

[0044] Fermentation time: Furthermore, it is preferred to add natural lignocellulosic biomass 16 hours after fermentation.

[0045] Furthermore, after the co-culture system is fermented, the supernatant of the fermentation liquid is collected to separate vanillin.

[0046] Beneficial effects

[0047] The present invention provides a co-cultivation method, wherein a recombinant strain capable of hydrolyzing xylan to release ferulic acid and a recombinant strain capable of using ferulic acid to produce vanillin are constructed. The co-cultivation of the two strains achieves vanillin biosynthesis using xylan as a substrate. The inventors further knocked out the genes for aldehyde dehydrogenases (ALDRs), alcohol dehydrogenases (ADHs), and aldehyde-keto reductases (AKRs) in the vanillin-synthesizing recombinant strain, thereby mitigating the conversion of vanillin into byproducts, vanillic acid and vanillyl alcohol, and effectively improving production efficiency. The advantages of this method are: (1) the existing technology requires the direct addition of high-cost ferulic acid as a substrate, which is relatively costly. The co-culture system in the present invention provides ferulic acid through in situ hydrolysis of xylan, which reduces the cost of raw materials and is suitable for large-scale production; (2) the existing technology directly uses ferulic acid as a substrate to synthesize vanillin. High concentrations of ferulic acid have an inhibitory effect on the activity of the strain, while the co-culture system in the present invention effectively alleviates the substrate toxicity problem by dynamically releasing ferulic acid; (3) a single-bacterial system simultaneously undertakes xylan hydrolysis and vanillin synthesis, which will lead to excessive metabolic burden, while co-culture improves the efficiency of the strain through the division of labor metabolic pathways, and the vanillin production reaches 0.7 mM within 72 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 : Determination of ferulic acid content in various pretreated natural substrates by alkaline hydrolysis method.

[0049] Figure 2 : Construction of recombinant plasmid pPICZαA-rXyn10A-Fae1A for xylan hydrolyzing strain XF.

[0050] Figure 3: Xylan hydrolysis ability and product analysis of the xylan hydrolyzing strain XF. a: Ferulic acid release from XF; b: Reducing sugar content of XF; c: Analysis of hydrolysis products from XF; d: Surface morphology and structure of the natural substrate under scanning electron microscopy; e: Fourier transform infrared spectroscopy analysis.

[0051] Figure 4 Optimization of natural substrate concentration during fermentation of the xylan hydrolyzing strain XF. a: Biomass of strain XF at different natural substrate concentrations; b: Ferulic acid release from strain XF at different natural substrate concentrations; c: Reducing sugar content of strain XF at different natural substrate concentrations.

[0052] Figure 5 :Construction of vanillin synthesis strain. a:Plasmid pPIC3.5K-ech R37M -fcs construction; b: blocking the vanillin conversion pathway of EF strain; c: ferulic acid conversion ability of EF strain and analysis of fermentation products.

[0053] Figure 6 Optimization of the inoculation ratio of different recombinant strains in the co-culture system. a: Vanillin titer of co-culture systems with different inoculation ratios; b: Reducing sugar titer of co-culture systems with different inoculation ratios; c: Ferulic acid release of co-culture systems with different inoculation ratios.

[0054] Figure 7 : Optimization of carbon sources in the co-culture system. a: Vanillin titer in co-culture systems with different carbon sources; b: Reducing sugar titer in co-culture systems with different carbon sources; c: Ferulic acid release in co-culture systems with different carbon sources.

[0055] Figure 8 :Schematic diagram of the co-culture system DETAILED DESCRIPTION

[0056] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the scope of protection of the present invention is not limited thereto. In the following examples, various processes and methods not described in detail are conventional methods well known in the art. The sources, trade names, and components of the reagents used are indicated when they first appear, and the same reagents used thereafter are the same as those indicated for the first time unless otherwise specified; the reagents, materials, etc. involved are all commercially available unless otherwise specified.

[0057] Pichia pastoris GS115 (our laboratory); ferulic acid and vanillin standards, and antibiotics (Solarbio, China); restriction enzymes (Beijing Langbolid Trading Co., Ltd.); agarose, nucleic acid electrophoresis dye, and DNAMaker (Beijing Bomade Gene Technology Co., Ltd.); plasmid miniprep kit and general DNA product purification kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.); tryptone and yeast extract (Oxoid, UK); the bifunctional enzyme xylanase / feruloyl esterase rXyn10A / Fae1A from the bacterial strain EMSD5, feruloyl-CoA synthase FCS from Amycolatopsis simulans, and the enoyl-CoA hydratase / aldolase mutant ECH from Amycolatopsis simulans. R37M Codon optimization and synthesis (BGI Genomics Co., Ltd.).

[0058] The culture medium involved in the following examples is as follows

[0059] YPD liquid medium: 20 g / L glucose, 20 g / L tryptone, 10 g / L yeast extract, sterilized at 115°C for 30 min.

[0060] YPD agar medium: Add 15g agar powder to 1L of YPD liquid medium, sterilize with high-pressure steam, and pour into sterile plates to obtain antibiotic-free YPD solid medium; if antibiotics are added, the corresponding antibiotic-selective YPD solid medium will be obtained.

[0061] The formula of BMGY medium is: 20g / L peptone, 3g / L K2HPO4, 11.8g / L KH2PO4, add water to 890mL, sterilize at 120℃ for 20min, and after cooling to 60℃, add 100mL of 10×YNB (13.4g / L), 2mL of 500×biotin (4×10-4g / L), and 10mL of glycerol in a clean bench.

[0062] The BMGY medium formula is: 20 g / L peptone, 3 g / L K2HPO4, 11.8 g / L KH2PO4, add water to 890 mL, sterilize at 120°C for 20 min, and after cooling to 60°C, add 10× YNB 100 mL (13.4 g / L), 500× biotin 2 mL (4×10-4 g / L), and methanol 5 mL in a clean bench.

[0063] LLB liquid medium: 5 g / L NaCl, 10 g / L tryptone, 5 g / L yeast extract, sterilized at 120°C for 20 min.

[0064] LLB agar medium: Add 15g agar powder to 1L of LLB liquid medium, sterilize with high-pressure steam, and pour into sterile plates to obtain antibiotic-free LLB solid medium; if antibiotics are added, it becomes the corresponding antibiotic-selective LLB solid medium.

[0065] In the above culture medium, the concentration of bleomycin in YPD medium is 50 μg / mL, the concentration in LLB medium is 25 μg / mL, the concentration of G418 in YPD medium is 800 μg / mL, and the concentration of ampicillin in LLB medium is 100 μg / mL.

[0066] The strains, plasmids and primers involved in the following examples are shown in Table 1-2:

[0067] Table 1: Strains and plasmids

[0068]

[0069] Table 2: Primer sequences

[0070]

[0071] Culture conditions:

[0072] Seed culture: A single clone of the Pichia pastoris strain was inoculated into YPD medium (containing 10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose) and cultured at 30°C and 220 rpm with shaking until the OD 600 Reach 2-6.

[0073] Fermentation culture: The overnight culture seed solution was inoculated into BMGY medium containing 20 g / L peptone, 3 g / L K2HPO4, 11.8 g / L KH2PO4, 100 mL 10× yeast nitrogen base (YNB), 2 mL 500× biotin (4×10-4 g / L) and 10 mL glycerol, and cultured under the same conditions until OD 600 Then the culture was kept at 4°C overnight, and the precipitated cells were resuspended in BMMY medium (adjusted to OD 600 =3-4) and continue shaking at 30°C, 220 rpm. BMMY medium formulation: 20 g / L peptone, 3 g / L K₂HPO₄, 11.8 g / L KH₂PO₄, 100 mL 10× YNB, 2 mL 500× biotin, and 5 mL methanol. Methanol was added every 24 hours to a final concentration of 1.5% to induce exogenous gene expression.

[0074] Co-cultivation: After each strain was grown in BMGY medium, it was resuspended and adjusted to the same OD with BMMY medium at a 1:1 volume ratio. 600 value.

[0075] Ferulic acid content determination: The release of ferulic acid from natural lignocellulosic biomass was determined using an alkaline hydrolysis method. Specifically, 0.005 g of substrate was weighed and mixed with 1 mL of 2 M NaOH. The mixture was shaken at room temperature for 5 hours. The pH was then adjusted to 6-7 with HCl, and the final volume was adjusted to 2 mL. The mixture was centrifuged at 12,000 × g for 10 minutes. The supernatant was collected and mixed with an equal volume of ethanol. The ferulic acid concentration was determined using high-performance liquid chromatography (HPLC).

[0076] Fermentation product analysis: Quantitative analysis of ferulic acid and vanillin: An Agilent 1220 HPLC system equipped with a variable wavelength detector (VWD) and a ZORBAX SB-C18 column (5 μm, 4.6×250 mm, Agilent) was used to detect the concentrations of ferulic acid and vanillin at a wavelength of 280 nm. Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in methanol. The injection volume was 10 μL, and the flow rate was 0.5 mL / min. Sample detection conditions were: 0 to 8.5 minutes, 60% mobile phase A; 9.5 to 18 minutes, 15% mobile phase A; 19 to 25 minutes, 60% mobile phase A.

[0077] Reducing sugar analysis: The reducing sugar content was determined using the 3,5-dinitrosalicylic acid (DNS) method, and the absorbance was measured at a wavelength of 540 nm using a UVmini-1280 spectrophotometer (Shimadzu, Japan).

[0078] Scanning electron microscopy (SEM) analysis: Sample processing: The surface morphology was analyzed using a JSM-7401 scanning electron microscope (HITACHI, Japan). The samples were gently washed three times with phosphate buffered saline (PBS), collected by centrifugation at 3,000×g for 10 minutes at 4°C, and then fixed with 3.5% glutaraldehyde solution at room temperature for 48 hours. The fixed samples were washed three times with PBS, each washing time exceeding 5 minutes, and then dehydrated in 30%, 50%, 60%, 70%, 80%, 90%, 95% and 100% ethanol solutions for 5 minutes each. After air drying, a 10 nm thick gold layer was sputtered on the surface of the natural lignocellulosic biomass sample using a magnetron sputtering coater (Model EIKO IB-5, Eiko Inc., Japan). Finally, the surface morphology of the sample was observed using a scanning electron microscope at an accelerating voltage of 10 kV.

[0079] Example 1: Selection of natural lignocellulosic biomass

[0080] For example, corn cobs, corn straw, rice straw, wheat straw, wheat bran and other agricultural by-products are selected as natural xylan substrates. The structure of natural lignocellulose is complex, and it is necessary to reduce the crystal structure of lignocellulose through pretreatment methods such as steam explosion and ultrafine grinding, increase the specific surface area of ​​the substrate, and improve the conversion efficiency of xylan. The total release of ferulic acid in different agricultural by-products after pretreatment was determined by alkaline hydrolysis. Among them, the ferulic acid release of ultrafine grinding corn cob powder (UGCC) was the highest, reaching 10.67 mg / g. The results are as follows Figure 1 shown.

[0081] Treatment method for destarched wheat bran (DSWB): soak 50 g of wheat bran in 500 mL of deionized water, add 1.5 g of α-amylase and incubate at 65°C for 30 min with intermittent stirring, then add 1.5 g of papain and incubate at 55°C for 45 min, then boil in water for 20 min to inactivate the enzyme solution, finally wash with deionized water until the supernatant is clear, filter, dry at 60°C and pass through a 50-mesh sieve, and store at 4°C for later use;

[0082] Corncob powder processing method: grind with a wall-breaking machine, pass through a 50-mesh sieve, and store at 4°C for later use;

[0083] Processing method of steam-exploded corn cobs (SECC) or steam-exploded corn straw (SECS): rehydrate 300 g of corn cobs or corn straw crushed to 50 mesh in 600 mL of deionized water for 12 hours, and then place the material into a steam explosion reactor. The steam explosion reactor consists of three parts: a steam generator, a reaction tank, and a receiving tank, which are regulated by a connected ball valve. The reactor volume is 2L. After preheating the reaction tank to 120°C, the rehydrated sample is introduced into the reaction tank. After maintaining a pressure of 0.8 (wheat bran) 1.0 (corn cob) MPa under saturated steam for 10 minutes, the discharge ball valve automatically opens and the material explodes. The steam-exploded material is collected in a receiving tank, washed with deionized water and the material in a ratio of 10:1 (w / v), taken out and dried in a blast drying oven. Store at 4°C for future use;

[0084] Processing method of ultrafinely ground corn cobs (UGCC), ultrafinely ground corn straw (UGCS), ultrafinely ground rice straw (UGRS), and ultrafinely ground wheat straw (UGWS): add the solids to the storage tank of the ultrafine grinder (YSC-715), process for 1 hour, and store at 4°C for use.

[0085] Example 2: Construction of upstream xylan hydrolysis module

[0086] The upstream xylan hydrolysis module XF strain was constructed using the bifunctional xylanase / feruloyl esterase (rXyn10A / Fae1A) from the EMSD5 bacterial consortium. This strain was constructed on the Pichia pastoris secretory expression vector pPICZαA, synthesized by Beijing Liuhe BGI Genomics Co., Ltd. and optimized according to the dominant codon usage of Pichia pastoris. The resulting recombinant plasmid, pPICZαA-rXyn10A-Fae1A (the nucleotide sequence of the introduced rXyn10A / Fae1A is SEQ ID NO: 20), was obtained. The recombinant plasmid pPICZαA-rXyn10A / Fae1A was linearized using the restriction endonuclease Sac I. The digestion results were verified by agarose gel electrophoresis, which showed that the linearized plasmid had a lower migration rate than the supercoiled plasmid. The linearized plasmid was then concentrated and electroporated, and screened on YPD plates containing 50 μg / mL zeocin resistance. Single colonies were picked and verified by colony PCR using primers rXyn10A / Fae1A-F and rXyn10A / Fae1A-R, and the target bands with the same size were sequenced to obtain the xylan hydrolysis strain XF. Figure 2 Shown is a plasmid construction diagram.

[0087] Example 3: Xylan hydrolysis ability and product analysis of xylan hydrolysis module strain XF

[0088] Fermentation was performed using 20 g / L UGCC as a substrate, and the amount of ferulic acid released by the xylan-hydrolyzing strain XF during fermentation at different fermentation times was determined using high-performance liquid chromatography. After 72 hours of fermentation, strain XF hydrolyzed UGCC to produce 0.58 mM ferulic acid, a yield of 5.62 mg / g substrate, representing 52.7% of the total alkali-extractable ferulic acid. Furthermore, the reducing sugar concentration in the fermentation broth reached 0.296 mg / mL at 72 hours of fermentation. To further explore the structural changes in the natural substrate during fermentation, scanning electron microscopy (SEM) was used to observe changes in the surface morphology of UGCC produced by strain XF at 0, 48, and 72 hours of fermentation. At the initial stage of fermentation (0 h), UGCC was a regular block structure with holes. As fermentation progressed, a large number of yeasts attached to the surface of UGCC. Compared with the WT control group, strain XF degraded the original relatively complete and dense UGCC into small pieces of loose structure at 48 h of fermentation, indicating that the xylan hydrolyzing strain XF can effectively hydrolyze UGCC. The results are shown in Figure 2. Figure 3 shown.

[0089] Example 4: Optimization of natural substrate concentration of xylan hydrolysis module strain XF

[0090] In a co-culture system, ferulic acid release directly affects vanillin production. Therefore, the UGCC substrate concentration was optimized to investigate the effects of 2% (20 g / L), 4% (40 g / L), 6% (60 g / L), 8% (80 g / L), and 10% (100 g / L) UGCC substrate concentrations on the biomass, ferulic acid production, and reducing sugar concentration of strain XF. Strain XF maintained normal growth at various substrate concentrations. At a UGCC concentration of 20 g / L, strain XF hydrolyzed xylan to produce 0.53 mM ferulic acid and 0.296 mg / mL reducing sugars (the main component being xylobiose). Ferulic acid release gradually increased with increasing substrate concentration, but the relationship was not linear. Ferulic acid release reached its peak at a UGCC concentration of 100 g / L, with 0.93 mM ferulic acid released and a reducing sugar concentration of 1.096 mg / mL after 48 hours of fermentation. Scanning electron microscopy (SEM) was used to observe the degree of cell wall damage, and combined with the changes in characteristic peaks of Fourier transform infrared spectroscopy (FTIR), it was confirmed that strain XF had a significant hydrolysis effect on UGCC. Figure 4 shown.

[0091] Example 5: Construction of downstream vanillin synthesis module

[0092] Using the ech of Amycolatopsis sp.ATCC 39116 R37M The mutant and fcs gene were used to construct the vanillin synthesis strain EF. The optimized nucleotide sequence was obtained by site-directed mutagenesis, as shown in SEQ ID NO: 18 and SEQ ID NO: 16. After sequence optimization according to the principle of Pichia pastoris dominant codon usage (SEQ ID NO: 19), it was synthesized by Beijing Liuhe BGI Genomics Co., Ltd. and constructed into the Pichia pastoris expression vector pPIC3.5K to obtain the recombinant plasmid pPIC3.5K-ech R37M The recombinant plasmid was linearized using the restriction endonuclease BspEⅠ, and the digestion results were verified by agarose gel electrophoresis (the migration rate of the linearized plasmid was slower than that of the supercoiled plasmid). Subsequently, the linearized plasmid was concentrated and electroporated into Pichia pastoris. Positive transformants were screened on YPD plates containing 800 μg / mL G418-resistant strains. Colony PCR and sequencing were performed using primers YAN-ech-F / R and YAN-fcs-F / R to obtain the vanillin-producing strain EF.

[0093] To inhibit the production of by-products such as vanillic acid and vanillyl alcohol, 15 genes encoding ADHs, AKRs and ALDRs (SEQ ID NOs: 1-15) in the Pichia pastoris genome were identified. The genes encoding aldehyde dehydrogenases (ALDRs) PAS_chr3_0987, PAS_chr2-1_0453, PAS_chr4_0043, PAS_chr4_0470, PAS_chr2-1_0853, alcohol dehydrogenases (ADHs) PAS_chr3_0006, PAS_chr4_0576, PAS_chr1-1_0357, PAS_chr2-1_0313, PAS_chr2-1_0472, PAS_chr3_1028, and aldehyde-keto reductases (AKRs) PAS_chr1-1_0360, PAS_chr2-1_0573, PAS_chr4_0336, PAS_chr3_0446 in Pichia pastoris were knocked out. The vanillin biosynthesis capacity of strain EF was determined by HPLC using 10 mM ferulic acid as substrate. Strain EF completely consumed the substrate within 48 h, and the vanillin yield after 36 h of fermentation was 9.56 mM, with a molar conversion rate of 95.6%. Figure 5 shown.

[0094] Example 6: Comparative verification of different gene knockout strategies

[0095] This example is used to verify the comparison of different gene knockout strategies

[0096] Gene knockout method: Using PAS_chr3_0987 as an example, we first obtained the upstream and downstream homology arm sequences (1000 bp) of PAS_chr3_0987 from NCBI. PCR was then performed to generate the fragments UP-PAS_chr3_0987 and DN-UP-PAS_chr3_0987. Overlap PCR was then performed to generate the donor fragment UP-PAS_chr3_0987 + DN-UP-PAS_chr3_0987 (2000 bp). The sgRNA sequence for PAS_chr3_0987 was obtained from the online sgRNA design website chopchop (http: / / chopchop.cbu.uib.no / ), and the sgRNA plasmid sgRNA-PAS_chr3_0987 was obtained using Quik Change. The donor fragment and sgRNA plasmid were electroporated into Pichia pastoris, and the PAS_chr3_0987 knockout strain was obtained by PCR verification and plasmid elimination in YPD liquid.

[0097] The above method was used to perform gene knockout of different types in multiple groups, and recombinant Pichia pastoris of experimental groups 1-4 was constructed. The production of vanillin, vanillic acid, and vanillyl alcohol was detected by high performance liquid chromatography using 10 mM ferulic acid as a substrate.

[0098]

[0099]

[0100] The results showed (see Figure 5 b) The vanillin accumulation results of experimental group 4 (recombinant Pichia pastoris with simultaneous knockout of alcohol dehydrogenases (ADHs), aldehyde dehydrogenases (ALDRs), and aldehyde-keto reductases (AKRs)) exceeded those of experimental groups 1, 2, and 3 without gene knockout, as well as the strains with individual gene knockouts. This suggests that multiple gene knockouts can produce a synergistic effect.

[0101] Example 7: Optimization of inoculation ratios of different modules in the co-culture system

[0102] Using 100 g / LU GCC as the substrate, the xylan-hydrolyzing strain XF and the vanillin-producing strain EF were co-cultured at initial inoculum ratios of 1:1, 2:1, 3:1, 5:1, 10:1, 20:1, and 50:1, respectively. Accumulation of the intermediate ferulic acid was detected in all groups except the 1:1 inoculum. After 72h of fermentation, the vanillin yield of the group with an inoculation ratio of 1:1 was the highest, at 0.34mM (molar conversion rate of 36.6%); the vanillin yield of the group with an inoculation ratio of 2:1 was 0.23mM (molar conversion rate of 24.7%), the vanillin yield of the group with an inoculation ratio of 3:1 was 0.31mM (molar conversion rate of 33.3%), the vanillin yield of the group with an inoculation ratio of 5:1 was 0.23mM (molar conversion rate of 24.7%), the vanillin yield of the group with an inoculation ratio of 10:1 was 0.22mM (molar conversion rate of 23.7%), the vanillin yield of the group with an inoculation ratio of 20:1 was 0.26mM (molar conversion rate of 30%), and the vanillin yield of the group with an inoculation ratio of 50:1 was 0.21mM (molar conversion rate of 22.6%). The reducing sugar content produced was determined using the DNS method. At 72h, the reducing sugar content of the group with an inoculation ratio of 1:1 was the highest (0.61mg / mL reducing sugar). Figure 6 .

[0103] Example 8: Optimization of different carbon sources in co-cultivation system

[0104] The effects of three mixed carbon sources, methanol-glycerol, methanol-glucose and methanol-sorbitol (the ratio of methanol to other carbon sources is 1:1) on the synthesis of vanillin in the co-culture system were compared. 100g / L UGCC was used as the substrate, strain XF and strain EF were inoculated at a ratio of 1:1, and a single methanol carbon source was used as the control group. The results showed that after 72h of fermentation, the vanillin production of the methanol control group was 0.37mM (molar conversion rate was 39.8%), and the methanol-glucose group and the methanol-sorbitol group were slightly higher than the control group, both at 0.49mM (molar conversion rate was 52.6%). The vanillin production of the methanol-glycerol group was significantly higher than that of the control group, reaching 0.70mM, an increase of 51.4% over the control group, and the molar conversion rate was 75.3%. The DNS method was used to determine the content of reducing sugars produced. After 72h of fermentation, the methanol-glycerol group had the highest reducing sugar content (0.69mg / mL), further verifying its carbon source advantage. Figure 7 .

[0105] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A recombinant Pichia pastoris capable of converting ferulic acid into vanillin, characterized in that: One or more of the endogenous aldehyde dehydrogenase (ALDRs), alcohol dehydrogenase (ADHs), and aldehyde-keto reductase (AKRs) genes are knocked out.

2. The recombinant Pichia pastoris according to claim 1, wherein the knocked-out aldehyde dehydrogenase (ALDRs) encoding gene comprises at least one of PAS_chr3_0987, PAS_chr2-1_0453, PAS_chr4_0043, PAS_chr4_0470, and PAS_chr2-1_0853; the knocked-out alcohol dehydrogenase (ADHs) encoding gene comprises at least one of PAS_chr3_0006, PAS_chr2-1_0453, PAS_chr4_0043, PAS_chr4_0470, and PAS_chr2-1_0853; at least one of PAS_chr4_0576, PAS_chr1-1_0357, PAS_chr2-1_0313, PAS_chr2-1_0472, and PAS_chr3_1028; and the knocked-out aldehyde-ketone reductase (AKRs) encoding genes include at least one of PAS_chr1-1_0360, PAS_chr2-1_0573, PAS_chr4_0336, and PAS_chr3_0446.

3. The recombinant Pichia pastoris according to claim 2, wherein the knockout aldehyde dehydrogenase (ALDRs) encoding gene is at least one of SEQ ID NOs: 1-5; the knockout alcohol dehydrogenase (ADHs) encoding gene includes at least one of SEQ ID NOs: 6-11; and the knockout aldehyde-keto reductase (AKRs) encoding gene includes at least one of SEQ ID NOs: 12-15.

4. The recombinant Pichia pastoris according to any one of claims 1 to 3, further comprising heterologously expressing feruloyl-CoA synthase (FCS) and / or enoyl-CoA hydratase / aldolase (ECH) by genetic engineering methods.

5. The recombinant Pichia pastoris according to claim 4, wherein the amino acid sequence of the feruloyl-CoA synthase FCS is shown in SEQ ID NO: 23; and the amino acid sequence of the enoyl-CoA hydratase / aldolase ECH is shown in SEQ ID NO: 24 or SEQ ID NO:

21.

6. The recombinant Pichia pastoris according to claim 5, wherein the recombinant Pichia pastoris is obtained by converting FCS (SEQ ID NO: 16) and / or ECH / ECH R37M The nucleotide sequence (SEQ ID NO: 17 or 18) is integrated into the genome of the starting Pichia pastoris strain.

7. The recombinant Pichia pastoris according to claim 6, wherein GS115 is used as the starting strain, and the ECH R37M The nucleotide sequences of FCH and FCH were integrated into the His site on the genome of the starting strain GS115, and the genes of aldehyde dehydrogenases (ALDRs), alcohol dehydrogenases (ADHs) and aldehyde-keto reductases (AKRs) shown in SEQ ID NOs: 1-15 were further knocked out.

8. A co-cultivation system comprising the recombinant Pichia pastoris capable of converting ferulic acid to synthesize vanillin according to claims 1-7 and the recombinant Pichia pastoris capable of hydrolyzing xylan to release ferulic acid, for achieving the conversion of natural xylan substrate to vanillin.

9. The co-cultivation system according to claim 8, wherein the recombinant Pichia pastoris that hydrolyzes xylan to release ferulic acid is a recombinant Pichia pastoris that can overexpress the bifunctional enzyme xylanase / feruloyl esterase rXyn10A / Fae1A; further, the amino acid sequence of the bifunctional enzyme xylanase / feruloyl esterase rXyn10A / Fae1A is shown in SEQ ID NO:

22.

10. The co-cultivation system according to claim 9, wherein the recombinant Pichia pastoris that hydrolyzes xylan to release ferulic acid is obtained by introducing a recombinant strain comprising the nucleotide sequence of SEQ ID NO: 20 comprising the bifunctional xylanase / feruloyl esterase rXyn10A / Fae1A into Pichia pastoris.

11. The co-cultivation system according to claim 8, wherein the xylan is obtained by decomposing natural cellulose materials, including one or more of corn cobs, corn straw, rice straw, wheat straw, and wheat bran.

12. The co-cultivation system according to claim 11, wherein the natural cellulose material is treated by at least one treatment method selected from the group consisting of steam explosion, ultrafine grinding, ultrasound, machine grinding, acid hydrolysis, enzymatic hydrolysis, alkaline hydrolysis, organic solvents, and microbial degradation.

13. The co-cultivation system according to claim 11, wherein the concentration of the natural xylan substrate is 2%-10%, preferably 2%, 4%, 6%, 8% and 10%, and can also be 20g / L-100g / L, preferably 20g / L, 40g / L, 60g / L, 80g / L and 100g / L.

14. The co-cultivation system according to claim 11, wherein the seed liquid of the "recombinant Pichia pastoris capable of hydrolyzing xylan to release ferulic acid" and the "recombinant Pichia pastoris capable of converting ferulic acid to synthesize vanillin" are mixed in a ratio of (1-50):1 and inoculated into a Pichia pastoris culture medium for fermentation; preferably, the inoculation ratio can be 1:1, 2:1, 3:1, 5:1, 10:1, 20:1, 30:1, or 50:

1. The co-cultivation system according to claim 14 , wherein the carbon source used in the culture medium of the co-cultivation system is at least one of methanol, glycerol, glucose and sorbitol.

16. A co-cultivation method, comprising: inoculating a recombinant Pichia pastoris capable of hydrolyzing xylan to release ferulic acid and a recombinant Pichia pastoris capable of converting ferulic acid to synthesize vanillin at a ratio of 1:1 into a methanol-glycerol medium and fermenting for 48-72 hours; adding pretreated natural lignocellulosic biomass 12-16 hours into the fermentation process, and inducing the culture by adding methanol at a final concentration of 0.5%-1.5% every 24 hours; further, the recombinant Pichia pastoris capable of hydrolyzing xylan to release ferulic acid is a recombinant Pichia pastoris capable of overexpressing the bifunctional enzyme xylanase / feruloyl esterase rXyn10A / Fae1A, and the recombinant Pichia pastoris capable of converting ferulic acid to synthesize vanillin is the recombinant Pichia pastoris described in claims 1-7.

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