Method for synthesizing vanillin by synergistically converting lignocellulose

Abstract

The invention relates to the technical field of biology, in particular to a method for synthesizing vanillin through synergistic conversion of lignocellulose. The invention provides a genetic engineering strain and a new way, the enhanced conversion of a sugar way and the broadening of a substrate spectrum are realized, glucose, xylose, p-hydroxybenzoic acid, p-hydroxybenzaldehyde, p-coumaric acid, ferulic acid and vanillic acid are all synergistically converted to synthesize vanillin, in-situ product removal is performed through glycosylation, further enhanced synthesis is performed, and the yield of vanillin is increased. Therefore, a universal strategy is provided for realizing high-value utilization of all components of lignocellulose through a biotransformation method.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a method for synergistically transforming lignocellulose to synthesize vanillin. Background Art

[0002] Lignocellulosic biomass is the most abundant renewable raw material on Earth that can be used to produce biofuels and other chemical products. It is mainly composed of three components: cellulose, hemicellulose, and lignin. Cellulose accounts for 40% to 60% of lignocellulosic biomass and can be broken down into glucose, which is an ideal raw material for microbial growth and the synthesis of valuable products in biorefineries. Hemicellulose can also be used and converted by microorganisms after being hydrolyzed into xylose by dilute acid, alkali and specific enzymes. Traditionally, the utilization of the three major components of lignocellulosic is classified. For example, when cellulose biomass is utilized, the lignin component is often removed as a by-product, resulting in a waste of resources. Therefore, it is of great significance to convert all available monomers derived from the three components of lignocellulosic into high-value-added products. In addition, lignin is the largest renewable aromatic resource for the sustainable production of various chemicals, fuels and materials, which promotes the development of atomic economy. Previous studies tended to convert lignin-derived monomers, but cellulose is the most abundant component in lignocellulosic biomass and should be converted more efficiently. The synergistic utilization of all derived monomers of the three components is of great significance to achieve high-value conversion of lignocellulosic biomass.

[0003] Vanillin (also known as vanillin) is one of the most widely used spices in the world and is known as the "king of spices". The traditional production method of natural vanillin is plant extraction. This method is very expensive and its output is limited by climate, region and other factors. It cannot meet the world market demand for natural vanillin. Vanillin synthesized by microbial transformation is also natural vanillin or bio-vanillin and is a potential alternative to plant extraction. Aromatic compound monomers have a high conversion efficiency as raw materials, but pure precursors are often expensive and difficult to obtain, which also becomes a constraint on their production. The three major components of lignocellulose can be depolymerized into glucose, xylose and a variety of aromatic compound monomers, namely p-coumaric acid, ferulic acid, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, vanillic acid, etc. Current studies have shown that by constructing a brewer's yeast cell factory, glucose, xylose, p-coumaric acid, ferulic acid and p-hydroxybenzaldehyde can be converted into vanillin (patent application number CN202310747286.9), but the utilization rate of derivative monomers is not high enough and the types of available substrates are small, which is not conducive to the high value of all components of lignocellulose and does not meet bioeconomic requirements. Summary of the invention

[0004] In view of this, the present invention provides a method for the synergistic transformation of lignocellulose to synthesize vanillin. With the help of synthetic biology, genetic engineering and metabolic engineering, a new pathway for the synergistic transformation of lignocellulose, glucose, xylose, p-coumaric acid, ferulic acid, p-hydroxybenzoic acid, p-hydroxybenzaldehyde and vanillic acid is provided. A strategy for realizing the high value of all components of lignocellulose by utilizing the biological funnel pathway is also provided.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a genetically engineered bacterium, which uses Saccharomyces cerevisiae SyBE_Sc05220018 as a chassis, knocks out Aro7 and PGM1, and overexpresses Aro2 and Aro3 K222L and Aro4 K229L , and express UbiC, EcAROL, tkta, ppsa, SehpaC-(GGGGS)3-PahpaB, Pcg-1, CAR, SAH1, MET6, MET13atsc, XKS1, RsXI, and two copies of NtCOMT.

[0007] In some specific embodiments of the present invention, the genetically engineered bacteria uses Saccharomyces cerevisiae as the SyBE_Sc05220018 chassis, knocks out Aro7 and PGM1, integrates the UbiC expression cassette into YNRCdelta9, integrates the EcAROL expression cassette into TKL2-TEF2, integrates the tkta expression cassette into DAK2-ZNF1, integrates the ppsa expression cassette into DAK2-ZNF1, integrates the Aro2 expression cassette into DAK2-ZNF1, and integrates the Aro3 expression cassette into YNRCdelta9. K222L The expression cassette was integrated into DAK2-ZNF1, and Aro4 K229L The expression cassette was integrated into DAK2-ZNF1, the SehpaC-(GGGGS)3-PahpaB expression module was integrated into YPRCdelta15, the Pcg-1 expression cassette was integrated into YORWdelta22, the CAR expression cassette was integrated into YORWdelta22, two copies of the NtCOMT expression cassette were integrated into PDC5, the SAH1 expression cassette was integrated into HO, the MET6 expression cassette was integrated into HO, the MET13atsc expression cassette was integrated into HO, the XKS1 expression cassette was integrated into YDR541C, and the RsXI expression cassette was integrated into YDR541C.

[0008] In some specific embodiments of the present invention, the UbiC of the genetically engineered bacteria is derived from Escherichia coli; and / or

[0009] The EcAROL is derived from Escherichia coli; and / or

[0010] The TKTA is derived from Escherichia coli; and / or

[0011] The ppsa is derived from Escherichia coli; and / or

[0012] The XKS1 is derived from Saccharomyces cerevisiae; and / or

[0013] The RsXI is derived from termites.

[0014] In some specific embodiments of the present invention, the genetically engineered bacteria further have expression modules of 4CL and ECH;

[0015] The expression module of 4CL and ECH is: terminator-4CL-bidirectional inducible promoter-ECH-terminator.

[0016] In some specific embodiments of the present invention, the downstream 528 bp to 563 bp of APR7 of the genetically engineered bacteria are replaced with the expression modules of 4CL and ECH.

[0017] In some specific embodiments of the present invention, the genetically engineered bacteria also express UGT72B1 and complement PGM1.

[0018] In some specific embodiments of the present invention, the UGT72B1 of the above-mentioned genetically engineered bacteria is derived from Arabidopsis thaliana.

[0019] In some specific embodiments of the present invention, the genetically engineered bacteria integrate the expression cassette of UGT72B1 into EXG1.

[0020] In some specific embodiments of the present invention, the genetically engineered bacteria complement the PGM1 expression cassette back to its original position.

[0021] The present invention also provides the use of the genetically engineered bacteria in the preparation of vanillin.

[0022] The present invention also provides a method for preparing the above-mentioned genetically engineered bacteria, based on the preparation method of the genetically engineered bacteria containing Aro7, PGM1, Aro2, Aro3 K222L 、Aro4 K229L , UbiC, EcAROL, tkta, ppsa, SehpaC-(GGGGS)3-PahpaB, Pcg-1, CAR, SAH1, MET6, MET13atsc, XKS1, RsXI, NtCOMT;

[0023] The expression module may include at least one of the following:

[0024] Expression cassette with UbiC; Expression cassette with EcAROL; Expression cassette with ppsA, tktA, Aro2; Expression cassette with Aro3K222L 、Aro4 K229L An expression cassette; an expression cassette with SehpaC-(GGGGS)3-PahpaB; an expression cassette with Pcg-1 and CAR; an expression cassette with two copies of NtCOMT; an expression cassette with SAH1, MET6, and MET13atsc; an expression cassette with XKS1 and RsXI.

[0025] In some specific embodiments of the present invention, the method for preparing the genetically engineered bacteria comprises integrating the UbiC expression cassette into YNRCdelta9, integrating the EcAROL expression cassette into TKL2-TEF2, integrating the tkta expression cassette into DAK2-ZNF1, integrating the ppsa expression cassette into DAK2-ZNF1, integrating the Aro2 expression cassette into DAK2-ZNF1, and integrating the Aro3 K222L The expression cassette was integrated into DAK2-ZNF1, and Aro4 K229L The expression cassette was integrated into DAK2-ZNF1, the SehpaC-(GGGGS)3-PahpaB expression module was integrated into YPRCdelta15, the Pcg-1 expression cassette was integrated into YORWdelta22, the CAR expression cassette was integrated into YORWdelta22, two copies of the NtCOMT expression cassette were integrated into PDC5, the SAH1 expression cassette was integrated into HO, the MET6 expression cassette was integrated into HO, the MET13atsc expression cassette was integrated into HO, the XKS1 expression cassette was integrated into YDR541C, and the RsXI expression cassette was integrated into YDR541C.

[0026] In some specific embodiments of the present invention, the method for preparing the above-mentioned genetically engineered bacteria is based on an expression module containing at least one of 4CL and ECH.

[0027] In some specific embodiments of the present invention, the expression module of the method for preparing the genetically engineered bacteria may also include an expression module containing 4CL and ECH.

[0028] In some specific embodiments of the present invention, the method for preparing the genetically engineered bacteria comprises the step of replacing 528 bp to 563 bp downstream of APR7 with the expression modules of 4CL and ECH.

[0029] In some specific embodiments of the present invention, the method for preparing the above-mentioned genetically engineered bacteria is based on an expression module containing UGT72B1.

[0030] In some specific embodiments of the present invention, the method for preparing the above-mentioned genetically engineered bacteria includes integrating the UGT72B1 expression cassette into EXG1.

[0031] The present invention also provides a method for preparing vanillin, which is prepared based on the above genetically engineered bacteria.

[0032] In some specific embodiments of the present invention, the method for preparing vanillin comprises mixing the genetically engineered bacteria, culture medium, and substrate, culturing, and obtaining vanillin;

[0033] The substrate may be at least one of glucose, xylose, p-hydroxybenzoic acid, p-hydroxybenzaldehyde, p-coumaric acid, ferulic acid and vanillic acid.

[0034] The strain and the vanillin synthesis method of the present invention have the following effects:

[0035] The present invention provides a new approach to achieve enhanced conversion of the sugar pathway and broaden the substrate spectrum, and synergistically converts glucose, xylose, p-hydroxybenzoic acid, p-hydroxybenzaldehyde, p-coumaric acid, ferulic acid and vanillic acid into vanillin, and further enhances the synthesis by in situ product removal through glycosylation, thereby providing a universal strategy for achieving high-value utilization of all components of lignocellulose through bioconversion.

[0036] The chassis used in the present invention is Saccharomyces cerevisiae. Compared with other chassis microorganisms, Saccharomyces cerevisiae has biological safety and good robustness, a clear genetic background, mature molecular manipulation technology, and is convenient for the introduction of exogenous pathways and metabolic regulation. In addition, Saccharomyces cerevisiae is easy to culture at high density, which can effectively alleviate the problem of substrate and product inhibition, and improve the expression amount and catalytic efficiency of inefficient enzymes. As a recognized safe strain, Saccharomyces cerevisiae has better biological safety than other lignin-degrading bacteria, and is easier to implement subsequent optimization processes.

[0037] Compared with the published vanillin biosynthesis method, the present invention further broadens the achievable substrate spectrum within the same strain, providing more reference significance and research targets for vanillin biosynthesis.

[0038] The in-situ removal strategy of the glycosylated product of the present invention effectively alleviates the product inhibition problem and breaks through the main limiting factor of the bioconversion method for synthesizing vanillin.

[0039] Compared with the traditional method of utilizing wood cellulose, the present invention realizes the co-utilization of the three major components, converts all the derived monomers, reduces waste, is more in line with bio-economics, and also reduces the generation of waste. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0041] Figure 1The synthesis of p-hydroxybenzoic acid using glucose as substrate is shown in Figure 1, where a is a schematic diagram of the design and enhancement of the transformation pathway, b is a schematic diagram of the knockout or integration sites of exogenous and endogenous genes, and c is the concentration of vanillin synthesized by each strain in a 2-mL YPD fermentation system;

[0042] Figure 2 The results show that p-hydroxybenzoic acid was synthesized by using glucose as substrate in a shake flask, wherein a shows a fermentation time of 96 h, and b shows a fermentation time of 120 h;

[0043] Figure 3 The design of a pathway for the conversion of p-hydroxybenzoic acid into vanillin is shown;

[0044] Figure 4 It is an integrated schematic diagram of the downstream pathway of p-hydroxybenzoic acid;

[0045] Figure 5 The invention shows the conversion of p-hydroxybenzaldehyde and p-hydroxybenzoic acid into vanillin, wherein a shows the conversion of p-hydroxybenzaldehyde into vanillin, and b shows the conversion of p-hydroxybenzoic acid into vanillin;

[0046] Figure 6 It is shown that yScXIN392 synthesizes vanillin;

[0047] Figure 7 It is shown that yScXIN402 synthesizes vanillin;

[0048] Figure 8 The design of a pathway for the synthesis of vanillin from p-coumaric acid and ferulic acid is shown;

[0049] Fig. 9 The promoter design controls the orderly expression of 4CL and ECH;

[0050] Fig.10 The promoter design and fermentation optimization are shown to achieve multi-component co-transformation synthesis of vanillin, where a, b, and c respectively show three different expression modes;

[0051] Fig.11 The results showed that glycosylation could improve the conversion yield of all components. DETAILED DESCRIPTION

[0052] The present invention discloses a method for synthesizing vanillin by synergistic conversion of lignocellulose. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0053] The present invention uses the yeast strain that can efficiently convert glucose to synthesize vanillin constructed in the early stage as the chassis, designs and constructs a new and efficient way to synthesize vanillin with glucose, xylose, p-hydroxybenzoic acid, p-hydroxybenzaldehyde, p-coumaric acid, ferulic acid and vanillic acid as precursors, and a biological funnel way to synthesize vanillin with the three major components of non-natural lignocellulose monomers: (a) constructing a xylose pathway; (b) realizing the efficient conversion of vanillic acid to synthesize vanillin; (c) optimizing the promoter design to control the conversion of p-coumaric acid and ferulic acid; (d) glycosylation to remove product inhibition. All of the above strategies combined with the previous inventions jointly achieve the purpose of converting all the main monomers obtained by pretreatment and enzymatic hydrolysis of corn stalks such as glucose, xylose, ferulic acid, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, vanillic acid and p-coumaric acid into vanillin. The present invention helps to establish a sustainable solution for the biosynthesis of vanillin and the high value of lignocellulose.

[0054] The sequence information involved in the present invention is as follows:

[0055] Optimization of RsXI gene codons TDH1 -XKS1-T PGK1 -P TDH3 -RsXI-T CYC1 Modules:

[0056]

[0057]

[0058] Promoter P 1GAL10 :

[0059]

[0060] Promoter P HSP30 :

[0061]

[0062] Optimized 4CL gene sequence:

[0063]

[0064] Optimized ECH gene sequence:

[0065]

[0066] Optimized UGT72B1 gene sequence:

[0067]

[0068]

[0069] Chorisate lyase encoding gene UbiC from Escherichia coli (after codon optimization):

[0070]

[0071] EcAroL, a gene encoding shikimate kinase from large intestine (after codon optimization):

[0072]

[0073] Transketolase encoding gene TktA (after codon optimization):

[0074]

[0075] Phosphoenolpyruvate synthase encoding gene PpsA (after codon optimization):

[0076]

[0077]

[0078] Endogenous mutant Aro3 K222L :

[0079]

[0080] Endogenous mutant Aro4 K229L :

[0081]

[0082] CAR (after codon optimization):

[0083]

[0084]

[0085] Pcg-1 (after codon optimization):

[0086]

[0087] SehpaC-(GGGGS)3-PahpaB (after codon optimization):

[0088]

[0089]

[0090] NtCOMT (after codon optimization):

[0091]

[0092] MET13atsc (after codon optimization):

[0093]

[0094] UGT72B1 (after codon optimization):

[0095]

[0096] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0097] Unless otherwise specified, the sequences of genes, polypeptides or proteins involved in the present invention have been disclosed in the prior art, and their sequences are not limited as long as they can achieve the expected functions.

[0098] The present invention will be further described below in conjunction with the embodiments.

[0099] Example 1: Obtaining a strain capable of efficiently converting glucose into vanillin

[0100] The base is a Saccharomyces cerevisiae strain that can stably accumulate vanillin, which was provided by Yuan Yingjin's research group and has the strain number SyBE_Sc05220018 (i.e., strain ZR05 recorded in patent CN113201465A).

[0101] (1) Construction and optimization of strains capable of synthesizing p-hydroxybenzoic acid using glucose as a substrate

[0102] like Figure 1 As shown in a, the endogenous phosphoglycerate mutase encoding gene PGM1 (SGD: S000001610) of Saccharomyces cerevisiae was knocked out in strain SyBE_Sc05220018 to reduce the loss of glucose-6-phosphate G6P; the chorismate mutase encoding gene Aro7 (SGD: S000006264) was knocked out to block the conversion of chorismate CHA into prephenate PPA, and then the chorismate lyase encoding gene UbiC from Escherichia coli was introduced, that is, the integrated expression module P was replaced at the YNRCdelta9 (SGD: S000007145) site of the genome. TEF2 -UbiC-T ADH2 , and obtained strain yScXIN329, which was able to synthesize 103.0 mg / L and 195.5 mg / L vanillin in a 2-mL YPD fermentation system after 24 h and 48 h, respectively ( Figure 1 b and c in the figure).

[0103] In order to further strengthen the supply of shikimate pathway precursors and relieve the feedback inhibition of DAHP (3-deoxy-D-arabino-2-heptulosonic acid 7-phosphate) synthases Aro3 and Aro4 by tyrosine or phenylalanine, shikimate kinase EcAroL, transketolase TktA and phosphoenolpyruvate synthase PpsA from Escherichia coli were introduced into strain yScXIN329, and endogenous chorismate synthase Aro2 and DAHP synthase anti-feedback inhibition mutant Aro3 were overexpressed. K222L and Aro4 K229L The specific integration method and the synthesis of p-hydroxybenzoic acid by the obtained strain are as follows Figure 1 As shown in b and c. Replace the integrated expression module P at the TKL2-TEF2 (SGD: S000000321; SGD: S000000322) site ENO2 -EcAROL-T CYC1 , strain yScXIN332 was obtained, which could synthesize 160.0 mg / L and 255.5 mg / L vanillin in 2-mL YPD fermentation system after 24 h and 48 h, respectively; the integrated expression module P was replaced at the DAK2-ZNF1 (SGD: S000001841; SGD: S000001842) site. FBA1 -ppsA-T PGI1 -P PGK1 -tktA-T HXT7 -T TDH1 -P TEF2 -Aro2-T ADH2 The strain yScXIN348 was obtained, which could synthesize 170.0 mg / L and 267.0 mg / L vanillin in a 2-mL YPD fermentation system after 24 h and 48 h, respectively; the integrated expression module P was further replaced at the DAK2-ZNF1 site. ENO2 -Aro3 K222L -T CYC1 -P PDC1 -Aro4 K229L -T TDH3 , and obtained strain yScXIN349, which was able to synthesize 291.5 mg / L and 351.5 mg / L vanillin in a 2-mL YPD fermentation system after 24 h and 48 h, respectively.

[0104] In order to further explore the amount of precursors that the above-mentioned construction pathway can provide for vanillin synthesis, strain yScXIN349 was fermented in a 250-mL shake flask with 50 mL YPD system. After 96 h, 528.0 mg / L p-hydroxybenzoic acid (such as Figure 2By adding about 10 g / L of glucose substrate to the system every 24 h, after 120 h of fermentation, 1059.5 mg / L of p-hydroxybenzoic acid can be detected to be synthesized in the system (as shown in a in FIG. Figure 2 As shown in b). It can be seen that this approach has the potential to efficiently synthesize vanillin.

[0105] (2) Construction and verification of a strain capable of converting p-hydroxybenzoic acid into vanillin

[0106] The strain yScXIN349 obtained by the above construction can synthesize p-hydroxybenzoic acid from glucose. Subsequently, a carboxylic acid reduction process needs to be introduced to convert p-hydroxybenzoic acid into p-hydroxybenzaldehyde, which is then hydroxylated to generate protocatechuic aldehyde, and further methylated to achieve the final synthesis of vanillin. Figure 3 shown.

[0107] By screening the enzymes of the downstream pathway and optimizing the integration sites, the construction scheme of the downstream pathway was finally determined, such as Figure 4 Shown: Replacement of the integrated expression module P at the YPRCdelta15 site PGK1 -SehpaC-(GGGGS)3-PahpaB-T HXT7 , replacing the integrated expression module P at the YORWdelta22 site PGK1 -Pcg-1-T HXT7 -P TDH3 -CAR-T TDH1 , replace the integrated expression module T at the PDC5 site GPD1 -NtCOMT-P TDH3 -P TDH3 -NtCOMT-T GPD1 , replace the integrated expression module P at the HO site TDH3 -SAH1-T PGI1 -P GPM1 -MET6-T TEF1 -T CYC1 -P PDC1 -MET13atsc-T TDH3 , and finally obtained strain yScXIN392. Among them, SAH1 and MET6 are as described in patent application number CN202310747286.9 or paper https: / / doi.org / 10.1016 / j.cej.2024.149815.

[0108] (3) Characterization of the efficiency of synthesizing vanillin using p-hydroxybenzoic acid, p-hydroxybenzaldehyde and vanillic acid as precursors

[0109] In the vanillin synthesis pathway of strain yScXIN392, p-hydroxybenzoic acid and p-hydroxybenzaldehyde are both key intermediates. The efficiency of the strain in converting the two was verified. The results are as follows Figure 5 As shown, 3.78mmol / L p-hydroxybenzaldehyde can be converted into 1.37mmol / L vanillin, and 4.12mmol / L p-hydroxybenzoic acid can be converted into 1.04mmol / L vanillin.

[0110] The ability of the strain to convert vanillic acid into vanillin was further characterized. Figure 6 As shown, strain yScXIN392 has a high conversion efficiency of vanillic acid, and can synthesize 6.40 mmol / L vanillin within 48 hours. It can fully utilize 6.71 mmol / L vanillic acid within 96 hours. Combined with the part of glucose converted in YPD, a total of 7.10 mmol / L vanillin was synthesized, achieving the highest yield of vanillin biotransformation by Saccharomyces cerevisiae.

[0111] Example 2: Construction of a pathway for synthesizing vanillin from xylose

[0112] Based on the yScXIN392 strain, xylose isomerase from termites was introduced, that is, the YDR541C site (SGD: S000002949) was replaced by the integrated expression P TDH1 -XKS1-T PGK1 -P TDH3 -RsXI-T CYC1 Module (XKS1 sequence: https: / / www.yeastgenome.org / #:~:text=The%20Saccharomyces%20Genome%20Database%20(SGD)%20provides%20comprehensive%20integrated), and strain yScXIN402 was obtained.

[0113] The specific method is as follows: yeast promoter and terminator are obtained by PCR amplification, the gene fragment is synthesized by a biological company, and then the promoter, gene and terminator are sequentially connected into a complete expression module by Overlap PCR method, and sequence verification is performed; PCR amplification is used to obtain the left and right approximately 400 bp sequences of the endogenous YDR541C gene of Saccharomyces cerevisiae, named YDR541C-L and YDR541C-R, which are respectively linked to the promoter P TDH1 and terminator T CYC1 Overlap the sequences to obtain YDR541C-LP TDH1 and YDR541C-RT CYC1As the connecting homology arm (YDR541C-HAs). According to the YDR541C sequence, construct the gRNA plasmid (containing the gene encoding the cas9 protein), and connect the gRNA plasmid, YDR541C-Has, and module P TDH1 -XKS1-T PGK1 -P TDH3 -RsXI-T CYC1 The transformants were introduced into Saccharomyces cerevisiae yScXIN392 by lithium acetate transformation method, coated on SC screening plates, and the obtained transformants were streaked for pure culture. The yeast genome was extracted for PCR verification and sequencing. The verified correct transformant was named yScXIN402.

[0114] The fermentation was carried out in YPX medium (containing 10 g / L yeast extract, 20 g / L peptone and 10 g / L xylose). Figure 7 As shown, 16 mg / L vanillin was synthesized. 357.0 mg / L can be synthesized in YPD medium, and 423.7 mg / L vanillin can be synthesized in YPDX medium (containing 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose and 10 g / L xylose). It can be seen that the efficiency of xylose conversion by this pathway is not high, but it can promote the synthesis of vanillin in the process of co-utilization of glucose and xylose. The construction of the xylose conversion pathway can not only assist the synthesis of vanillin, but also serve as a supplementary carbon source in the subsequent conversion of p-coumaric acid and ferulic acid.

[0115] Example 3: Construction of a pathway for synthesizing vanillin using p-coumaric acid and ferulic acid as precursors

[0116] The strain yScXIN402 obtained above can convert glucose, xylose, p-hydroxybenzaldehyde, p-hydroxybenzoic acid and vanillic acid into vanillin. In order to minimize the impact of subsequent transformation on the existing performance of the strain, an inducible promoter is selected to control the expression of subsequent pathways. Figure 8 , the 528bp to 563bp sequence downstream of gene APR7 was replaced with the expression modules of 4CL and ECH to achieve the utilization of coumaric acid and ferulic acid. FBA1 , bidirectional inducible promoter P 1GAL10 and heat shock protein promoter P HSP30 , three expression patterns such as Fig. 9 As shown: (I) Using P FBA1 and T PGI1 Control ECH expression, P HSP30 and T GPM1 Control 4CL expression, that is, start the expression of ECH in the downstream at the beginning of fermentation, the upstream 4CL is weakly expressed in the glucose utilization stage, and when the glucose is consumed and the xylose utilization stage begins, 4CL is strongly expressed; (II) use P FBA1and T PGI1 Control ECH expression, P 1GAL10 and T GPM1 The expression of 4CL was controlled and the GAL80 gene was knocked out by CRISPR method, with the aim of starting ECH expression at the beginning of fermentation. The expression of upstream 4CL was completely inhibited in the presence of glucose. When glucose was consumed, 4CL was P 1GAL10 Rapid expression; (III) using P 1GAL10 Simultaneously control the expression of 4CL and ECH (terminators are T GPM1 and T PGI1 ), so that both are inhibited in the glucose conversion stage, and the expression is started at the same time after the glucose is consumed. Gene knockout and integrated expression are both completed by the CRISPR system, and the specific steps are the same as those described in Example 2. The obtained strains are yScXIN412, yScXIN413 and yScXIN414.

[0117] The fermentation of strains yScXIN412, yScXIN413 and yScXIN414 was characterized in a medium containing glucose, xylose, p-coumaric acid and ferulic acid, and the addition time of the two aromatic substrates was optimized. Fig.10 As shown, the expression pattern in strain yScXIN414 had the least effect on the sugar pathway, the addition of p-coumaric acid and ferulic acid at 48 h had the lowest inhibitory effect on the strain, and the maximum yield of vanillin synthesized by multi-substrate co-conversion was 472.0 mg / L, which was 1.5 times the fermentation yield when only glucose and xylose were used as substrates.

[0118] Example 4: The aldehydes, bean curd, aldehydes, and aromatic acids are produced by the removal of the aromatic acid and the conversion of glucose, xylose, p-hydroxybenzoic acid, and p-hydroxybenzoic acid.

[0119] Based on strain yScXIN414, the Arabidopsis thaliana-derived glycosyltransferase UGT72B1 was introduced to glycosylate vanillin, that is, the expression module P was expressed using the CRISPR system. TEF2 -UGT72B1-T ADH2 The endogenous glycoside hydrolase encoding gene EXG1 of Saccharomyces cerevisiae was integrated and replaced, and the PGM1 gene was expressed in situ to promote the conversion of G6P to glucose-1-phosphate G1P, thereby increasing the supply of UDP-glucose. The resulting strain was named yScXIN428. By adding 716.5 mg / L p-coumaric acid, 225.5 mg / L ferulic acid and 114.5 mg / L vanillic acid to the YPDX medium at 48 h of fermentation, combined with p-hydroxybenzoic acid and p-hydroxybenzaldehyde contained in the pathway, the strain yScXIN428 was able to synthesize 1745.4 mg / L glucovanillin, equivalent to 845.0 mg / L vanillin, and almost no accumulation of by-products and intermediates ( Fig.11 ).

[0120] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A genetically engineered bacterium, characterized in that: Using Saccharomyces cerevisiae SyBE_Sc05220018 as the chassis, Aro7 was knocked out and Aro2 and Aro3 were overexpressed K222L and Aro4 K229L , and express UbiC, EcAROL, tkta, ppsa, SehpaC-(GGGGS)3-PahpaB, Pcg-1, CAR, SAH1, MET6, MET13atsc, XKS1, RsXI, and two copies of NtCOMT.

2. The genetically engineered bacterium according to claim 1, characterized in that include: The UbiC is derived from Escherichia coli; and / or The EcAROL is derived from Escherichia coli; and / or The TKTA is derived from Escherichia coli; and / or The ppsa is derived from Escherichia coli; and / or The XKS1 is derived from Saccharomyces cerevisiae; and / or The RsXI is derived from termites.

3. The genetically engineered bacterium according to claim 1 or 2, characterized in that: It also has expression modules for 4CL and ECH; The gene elements included in the expression module of 4CL and ECH are terminator, 4CL, bidirectional inducible promoter, ECH and terminator in sequence.

4. The genetically engineered bacterium according to claim 3, characterized in that UGT72B1 is also expressed.

5. The genetically engineered bacterium according to claim 4, characterized in that The UGT72B1 is derived from Arabidopsis thaliana.

6. Use of the genetically engineered bacteria according to any one of claims 1 to 5 in the preparation of vanillin.

7. The method for preparing a genetically engineered bacterium according to claim 1 or 2, characterized in that: Based on Aro7, Aro2, Aro3 K222L 、Aro4 K229L , UbiC, EcAROL, tkta, ppsa, SehpaC-(GGGGS)3-PahpaB, Pcg-1, CAR, SAH1, MET6, MET13atsc, XKS1, RsXI, and NtCOMT.

8. The method for preparing a genetically engineered bacterium according to claim 3, characterized in that: Prepared based on an expression module containing at least one of 4CL and ECH.

9. The method for preparing a genetically engineered bacterium according to claim 4 or 5, characterized in that: Prepared based on an expression module containing UGT72B1.

10. A method for preparing vanillin, characterized in that: Prepared based on the genetically engineered bacteria according to any one of claims 1 to 5.

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