CN113234611B

CN113234611B - Saccharomyces cerevisiae engineering bacteria and application thereof in preparation of protocatechuic acid

Info

Publication number: CN113234611B
Application number: CN202110495688.5A
Authority: CN
Inventors: 元英进; 李炳志; 张仁宽
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2021-05-07
Filing date: 2021-05-07
Publication date: 2023-01-20
Anticipated expiration: 2041-05-07
Also published as: CN113234611A

Abstract

The invention relates to the technical field of genetic engineering, in particular to a saccharomyces cerevisiae engineering bacterium and application thereof in preparation of protocatechuic acid. The invention is based on a saccharomyces cerevisiae strain, and at least one of exogenous genes 4CL, ech, fcs, vdh and PobA is transferred. According to the invention, by accurately controlling the transfer of the key genes, a brand-new path for synthesizing the protocatechuic acid by converting lignin or p-coumaric acid is constructed in the yeast body, and the synthesis of the protocatechuic acid by using lignin monomers and lignin is realized. Experiments show that the genetically engineered bacteria can fully utilize lignin and aromatic compounds derived from the lignin to prepare protocatechuic acid, not only can solve the utilization problem of the lignin, but also can generate the protocatechuic acid which is an important chemical raw material to realize high-value utilization of the lignin.

Description

Saccharomyces cerevisiae engineering bacteria and application thereof in preparation of protocatechuic acid

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a saccharomyces cerevisiae engineering bacterium and application thereof in preparation of protocatechuic acid.

Background

Protocatechuic acid is used as medicine for treating chronic tracheitis, burn, infantile pneumonia, bacillary dysentery, acute pyelonephritis, acute pancreatitis and ulcer. In addition, it has certain antioxidant and antibacterial effects, and can inhibit Staphylococcus aureus, salmonella, escherichia coli, etc. Protocatechuic acid is a key metabolic intermediate, and can be further converted into a plurality of chemical substances, such as adipic acid, pyridine 2,4-dicarboxylic acid and the like. In addition, in recent studies, protocatechuic acid has been used as a monomer for synthesizing polymers. Therefore, protocatechuic acid has wide application prospect, the chemical synthesis mainly takes vanillin as raw material, the vanillin is subjected to alkali melt oxidation and demethylation, and then the vanillin is subjected to acidification preparation, the process needs high temperature of over 240 ℃, the reaction is solid-phase reaction in most time and is difficult to control, special reaction equipment is required, the investment is large, and the equipment utilization rate is low. The technology for synthesizing protocatechuic acid by microbial transformation has been developed and researched. Protocatechuic acid is mainly formed by acting 3-dehydroshikimic acid dehydratase (aroZ) on 3-dehydroshikimic acid (DHS) in one step through catalysis. The gene is utilized to synthesize protocatechuic acid from glucose in escherichia coli, bacillus, corynebacterium glutamicum and saccharomyces cerevisiae, and the method is environment-friendly and sustainable, and has economic competitiveness and good industrial application prospect compared with a chemical method. In addition to synthesizing protocatechuic acid from sugar, synthesizing protocatechuic acid from lignin can reduce the use of glucose, promote the utilization of biomass and reduce the pollution of agricultural wastes to the environment. The research and research currently aims at the lignin degradation microorganisms, such as rhodococcus, pseudomonas putida, converting lignin and synthesizing protocatechuic acid and derived molecules thereof. However, the lignin degradation bacteria can degrade protocatechuic acid, so that the protocatechuic acid cannot be accumulated, and measures such as gene knockout are required. The method for synthesizing the protocatechuic acid by converting lignin by using the biologically safe strain yeast saccharomyces cerevisiae is not researched, and aiming at the main monomer composition of lignin hydrolysate, the method for synthesizing the protocatechuic acid by constructing and optimizing the lignin monomer realizes the efficient synthesis of the protocatechuic acid by the saccharomyces cerevisiae.

Biosynthesis of protocatechuic acid is studied more frequently in terms of glucose, and introduction of ubic and pobA genes into E.coli synthesized protocatechuic acid at about 110mg/L using glucose. In addition, in Corynebacterium glutamicum, ubic gene was introduced, and 1140.0. + -. 11.6 mg/L) protocatechuic acid was obtained by fed batch fermentation in a fermenter. According to the method, the raw materials of the Saccharomyces cerevisiae, namely sucrose, are introduced into AroZ (DHS dehydroatase) and degron-tagged Aro1 are designed, so that the Saccharomyces cerevisiae CEN.PK synthesizes protocatechuic acid by utilizing the sucrose, and 5.6g/L protocatechuic acid is finally obtained by supplementing the sugar through the fermentation process and fermenting for 8 days. In addition to the synthesis of protocatechuic acid from sugars, starting from aromatic compounds without starting materials, there have also been studies in recent years. Dai Yujie, etc. invented a genetically engineered bacterium for preparing protocatechuic acid (3,4-dihydroxybenzoic acid) by using phenol as raw material and its construction method. The engineering bacteria contain p-hydroxybenzoic acid decarboxylase gene yclBCD and hydroxylase gene pobA, phenol is used as a raw material, the phenol is added with carboxyl by a biotransformation method to obtain p-hydroxybenzoic acid, and then the 3-position of the p-hydroxybenzoic acid is oxidized to obtain protocatechuic acid (3,4-dihydroxybenzoic acid). Yuan Jifeng and the like invent that PmLAAD, hmaS, HMO, BFD, HFD1, hpaBC and PobA are introduced into escherichia coli, and the synthesis of protocatechuic acid from tyrosine by the escherichia coli is realized. At present, no report of synthesizing protocatechuic acid by using lignin as a raw material is found.

Disclosure of Invention

In view of this, the invention provides a saccharomyces cerevisiae engineering bacterium and an application thereof in preparation of protocatechuic acid. The invention carries out gene modification on the basis of the saccharomyces cerevisiae strain, constructs a new lignin biotransformation path in a yeast body, and the obtained saccharomyces cerevisiae engineering bacteria can fully utilize lignin and aromatic compounds derived from the lignin to synthesize protocatechuic acid, thereby obviously improving the content of the protocatechuic acid.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a saccharomyces cerevisiae engineering bacterium, wherein a saccharomyces cerevisiae strain is used as a chassis strain and is transformed with at least one of exogenous genes 4CL, ech, fcs, vdh and PobA.

In the invention, the chassis strain takes a saccharomyces cerevisiae strain as a development strain, and ADH6, ADH7 and BDH2 genes are knocked out on the basis of the development strain. The starting strain can be any saccharomyces cerevisiae. In some embodiments, the starting strain is Saccharomyces cerevisiae strain BY4742. On the basis of a saccharomyces cerevisiae strain BY4742, knocking out genes ADH6, ADH7 and BDH2 to obtain a chassis strain, and naming the chassis strain as a strain ZR01.

The gene knockout method is not particularly limited, and the gene knockout method can be CRISPR technology or can utilize a yeast homologous recombination mechanism. In a specific embodiment of the invention, the genes ADH6, ADH7 and BDH2 are knocked out by CRISPR technology. The specific method comprises the following steps:

a400 bp sequence of Saccharomyces cerevisiae ADH6 gene (genbank No.: NM-001182831.3) was obtained BY PCR using Saccharomyces cerevisiae BY4742 as a template and designated ADH6L, ADH R, and was overlap to obtain ADH6-LR as donor DNA. Constructing gRNA plasmid according to ADH6 sequence, introducing gRNA, cas9 and donor DNA into Saccharomyces cerevisiae BY4742, coating SC screening plate, streaking, purifying and culturing the obtained transformant, extracting yeast genome, and performing PCR verification and sequencing. In the above manner, ADH7 (NM _ 001178812.1) and BDH2 (NM _ 001178203.1) are sequentially knocked out and named as ZR01.

In some embodiments, the Chassis strain ZR01 is transformed with any one of the foreign genes 1) to 5):

1) Ech and Fcs;

2) 4CL and Ech;

3) 4CL, ech and vdh;

4) 4CL, ech, vdh, and PobA;

5) vdh or PobA.

In the invention, the exogenous gene 4CL gene is derived from parsley. The Ech gene is derived from pseudomonas putida or streptomyces. The Fcs gene is derived from Pseudomonas putida or Streptomyces sp. The vdh gene and the pobA gene are derived from Pseudomonas putida.

The above foreign genes are all gene sequences known in the art, and all are full-length sequences.

In the specific embodiment of the present invention, those skilled in the art can also optimize the relevant sequences according to the codon preference of Saccharomyces cerevisiae. In the invention, the parsley-derived 4CL gene is an optimized sequence, and the sequence of the optimized 4CL gene is shown as SEQ ID NO:1 is shown.

In the invention, the Ech gene and the Fcs gene derived from Streptomyces sp.V-1 are derived from Streptomyces sp.V-1 strain, the accession number of the Ech gene is KC847406.1, and the accession number of the Fcs gene is KC847405.1. Ech gene (accession number AAN 68962.1), fcs gene (accession number AAN 68960.2), vdh gene (accession number AAN 68961.1) and PobA gene (accession number AAN 69138.1) derived from Pseudomonas putida (Pseudomonas putida) are specifically derived from Pseudomonas putida KT2440, and amplification primers for each gene are as follows:

Pp-FCS-F:5’-ATGAATAACGAAGCCCGCTCA-3’(SEQ ID NO：2)；

Pp-FCS-R:5’-TCAAGGCCGCACCTTGGC-3’(SEQ ID NO：3)；

Pp-ECH-F:5’-ATGAGCAAATACGAAGGCCG-3’(SEQ ID NO：4)；

Pp-ECH-R:5’-TCAGCGCTTGTAGGCCTGC-3’(SEQ ID NO：5)；

Pp-VDH-F:5’-ATGTTGCAGGTGCCTTTGCT-3’(SEQ ID NO：6)；

Pp-VDH-R:5’-CTAGATGGGATAGTGACGCGGG-3’(SEQ ID NO：7)；

Pp-PobA-F:5’-ATGAAAACTCAGGTTGCAATTATTG-3’(SEQ ID NO：8)；

Pp-PobA-R:5’-TCAGGCAACTTCCTCGAACG-3’(SEQ ID NO：9)。

in some embodiments, streptomyces-derived Ech (ssech) and Fcs (ssfcs) are transferred into the chassis strain ZR01 to obtain the engineered bacterium VAN1.

Transferring Ech (ppech) and Fcs (ppfcs) of pseudomonas putida in an Chassis strain ZR01 to obtain an engineering bacterium VAN2.

4Cl derived from parsley and Ech (ppech) derived from pseudomonas putida are transferred into the chassis strain ZR01 to obtain the engineering bacteria VAN3.

4Cl derived from parsley, ech (ppech) derived from pseudomonas putida and Vdh derived from pseudomonas putida are transferred into the chassis strain ZR01 to obtain the engineering bacteria VAC1.

Transferring 4Cl derived from parsley, ech (ppech), vdh and PobA derived from pseudomonas putida into a chassis strain ZR01 in the form of plasmid to obtain the engineering bacteria PCA1.

Further transferring into pRS413 plasmid on the basis of the strain PCA1 to obtain a strain PCA2.

In the Chassis strain ZR01, pobA derived from Pseudomonas putida was transferred to obtain a strain PCA3.

In the PCA 1-PCA 3 strains, foreign genes are introduced in the form of plasmids.

In the invention, 4Cl derived from parsley, ech (ppech), vdh and PobA derived from pseudomonas putida are integrated on a genome of ZR01 by utilizing a homologous recombination mechanism to obtain the engineering bacteria PCA4.

In the invention, the 5 'end of each gene in the endogenous gene and the exogenous gene is connected with a promoter, and the 3' end is connected with a terminator.

Wherein the promoter is a Saccharomyces cerevisiae endogenous promoter P _TPI1P 、P _FBA1 、P _ENO2 Or P _PGK1 。

The source of the terminator in the present invention is not particularly limited, and any terminator commonly used in the art may be used. In the invention, the terminator is an endogenous terminator of saccharomyces cerevisiae, in particular to T _TPI1P 、T _FBA1 、T _ENO2 、T _HXT7 、T _PGI1 、T _PGH Or T _GPM1 Specific types of terminators include, but are not limited to, these.

The invention also provides application of the saccharomyces cerevisiae engineering bacteria in preparation of protocatechuic acid.

The invention provides a construction method of saccharomyces cerevisiae engineering bacteria, which comprises the following steps: constructing an expression module of a promoter-exogenous gene/endogenous gene-terminator, and transforming the expression module and a linearized skeleton vector into a chassis strain.

In the invention, the transformation method is a lithium acetate transformation method.

The skeleton carrier is PRS415 or PRS413.

The invention also provides a preparation method of protocatechuic acid, which utilizes the saccharomyces cerevisiae engineering bacteria for fermentation.

In the present invention, the fermentation medium comprises coumarin acids or lignin.

According to the invention, on the basis of knocking out ADH6, ADH7 and BDH2 saccharomyces cerevisiae, at least one of exogenous genes 4CL, ech, fcs, vdh and PobA is transferred, a new lignin biotransformation path is constructed in a yeast body, and the path transforms lignin into p-hydroxybenzoic acid and protocatechuic acid by modifying key genes, so that the synthesis of protocatechuic acid by using lignin monomers and lignin is realized. Experiments show that the genetically engineered bacterium can fully utilize lignin and aromatic compounds derived from the lignin to prepare protocatechuic acid, not only can solve the utilization problem of the lignin, but also can generate the protocatechuic acid which is an important chemical raw material, and realize high-value utilization of the lignin.

Drawings

FIG. 1 is a schematic diagram showing the construction of engineered yeast strains VAN1, VAN2, VAN3 and VAC 1;

FIG. 2 shows the results of synthesizing p-hydroxybenzaldehyde and p-hydroxybenzaldehyde by converting lignin with strains VAN1, VAN2, VAN3 and VAC 1;

FIG. 3 shows the results of preparing protocatechuic acid by fermenting genetically engineered bacteria PCA2 and PCA 3;

FIG. 4 shows the results of producing protocatechuic acid from genetically engineered bacteria PCA4 with p-coumarinic acid of different concentrations as substrates;

FIG. 5 shows the result of producing protocatechuic acid by using pretreated corn stalk lignin as a substrate by using genetically engineered bacteria PCA4.

Detailed Description

The invention provides a saccharomyces cerevisiae engineering bacterium and application thereof in preparing protocatechuic acid. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is specifically noted that all such substitutions and modifications will be apparent to those skilled in the art and are intended to be included herein. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

The invention is further illustrated by the following examples:

example 1

1. Construction of strains VAN1 to VAN3

Amplification of Yeast promoter P by PCR _TPI1P Terminator T _GPM1 Respectively performing overlap with genes ssfcs (streptomyces-derived Fcs), ppfcs (Pseudomonas putida-derived Fcs) and 4CL to obtain a module P _TPI1P -ssfcs-T _GPM1 ，P _TPI1P -ppfcs-T _GPM1 ，P _TPI1P -4CL-T _GPM1 . Amplification of Yeast promoter P by PCR _FBA1 Terminator T _PGI1 Respectively carrying out overlap with genes ssech (Ech from streptomycete) and ppech (Ech from pseudomonas putida) to obtain a module P _FBA1 -ssech-T _PGI1 ,P _FBA1 -ppech-T _PGI1 . Cleavage of p with XhoI, bamHIrs415 plasmid, the linearized vector PRS415 is respectively connected with the module P _TPI1P -ssfcs-T _GPM1 ,P _FBA1 -ssech-T _PGI1 ；P _TPI1P -ppfcs-T _GPM1 ，P _FBA1 -ppech-T _PGI1 ；P _TPI1P -4CL-T _GPM1 ，P _FBA1 -ppech-T _PGI1 The saccharomyces cerevisiae ZR01 is introduced by a lithium acetate conversion method, the left and right homologous sequences of the vector are recombined with the vector and integrated on the vector, an SD-LEU solid plate (6.7 g/L of synthetic yeast nitrogen source YNB, 20g/L of glucose, 2g/L of leucine-deficient mixed amino acid powder and 2% of agar powder) is adopted for screening after conversion, the obtained transformant is subjected to streak purification culture, yeast genome is extracted for PCR verification, glycerol bacteria are respectively named as VAN1 VAN2 VAN3 for the recombinant strain with correct verification, and the construction schematic diagram is shown in figure 1.

2. Construction of the Strain VAC1

Building block T _PGI1 -P _ENO2 -vdh-T _ENO2 The linearized vector PRS415 is combined with the module P _TPI1P -4CL-T _GPM1 ，P _FBA1 -ppech-T _PGI1 ,T _PGI1 -P _ENO2 -vdh-T _ENO2 Saccharomyces cerevisiae ZR01 was transformed by the method of step 1 above to obtain VAC1, and the construction scheme is shown in FIG. 1.

3. Construction of strains PCA 2-PCA 4

Building block T _ENO2 -P _PGK1 -PobA-T _HXT7 The linearized carrier PRS415 is connected with the module P _TPI1P -4CL-T _GPM1 ，P _FBA1 -ppech-T _PGI1 ,T _PGI1 -P _ENO2 -vdh-T _ENO2 ,T _ENO2 -P _PGK1 -PobA-T _HXT7 Converting saccharomyces cerevisiae ZR01 by using the method in the step 1 to obtain PCA1; the pRS413 plasmid was further transformed into the PCA1 strain to obtain a PCA2 strain.

The linearized vector PRS413 is connected with the module T _ENO2 -P _PGK1 -PobA-T _HXT7 And (3) introducing the mixture into saccharomyces cerevisiae PCA1 by using the method in the step 1 to obtain PCA3.

Fragment P was converted by lithium acetate method _TPI1P -4CL-T _GPM1 ，P _FBA1 -ppech-T _PGI1 ,T _PGI1 -P _ENO2 -vdh-T _ENO2 ,T _ENO2 -P _PGK1 -PobA-T _HXT7 Transforming Saccharomyces cerevisiae ZR01, recombining left and right homologous sequences with HO site on yeast genome to integrate into genome, and converting module T _ENO2 -P _PGK1 -PobA-T _HXT7 The left and right homologous sequences are recombined with the digital 15 site on the yeast genome and integrated on the genome to obtain the stably inherited yeast strain PCA4.

Example 2 ability of different strains to synthesize protocatechuic acid

The strains VAN1, VAN2, VAN3, VAC1 constructed in example 1 and the Control strain Control (empty plasmid pRS415 transformed in ZR 01) were cultured for 24 hours in a SC-L medium initially supplemented with 200mg/L p-coumaric acid (synthetic yeast nitrogen source YNB 6.7g/L, glucose 20g/L, leucine-deficient mixed amino acid powder 2 g/L) to analyze the yields of p-hydroxybenzoic acid and p-hydroxybenzaldehyde, and the results are shown in FIG. 2.

The strains PCA2 and PCA3 constructed in example 1 were cultured in SC-Leu-His (synthetic yeast nitrogen source YNB 6.7g/L, glucose 20g/L, leucine-deficient mixed amino acid powder 2 g/L) medium initially supplemented with 200mg/L p-coumaric acid for 96 hours to analyze the yields of protocatechuic acid and p-hydroxybenzoic acid, and the results are shown in FIG. 3.

The strain PCA4 constructed in example 1 can produce 720mg/L of protocatechuic acid under the initial conditions of 200 (mg/L), 600 (mg/L), 1000 (mg/L), 1400 (mg/L), 1600 (mg/L), 2000 (mg/L) p-coumaric acid, and the result is shown in FIG. 4.

1.5g of sodium hydroxide is dissolved in 135mL of purified water, added to 15g of corn straw and placed in a reaction kettle, and subjected to oil bath at 130 ℃ for 30min. Cooling, filtering, adjusting pH to 6, and removing precipitate at 12000rpm 30min to obtain lignin hydrolysate.

The strain PCA4 constructed in example 1 was added to YPD medium of 0.5X lignin hydrolysate and fermented. The highest yield was 400mg/L protocatechuic acid, and the results are shown in FIG. 5.

As can be seen from the results in FIG. 2, the engineered yeast strains VAN1 VAN2, VAN3 and VAC1 constructed by the invention can convert lignin into protocatechuic acid precursor p-hydroxybenzoic acid, wherein the strain VAC1 with 4CL, ppEch and vdh transferred into has the best conversion effect, and the content of p-hydroxybenzoic acid is as high as 115mg/L.

FIG. 3 shows that the PCA2 strain was obtained by introducing pobA based on VAC1, and that 93mg/L of protocatechuic acid was synthesized using 200mg/L of p-coumaric acid. In order to further improve the protocatechuic acid yield and increase the copy number of pobA, the PCA4 strain obtains PCA4, the yield is obviously improved, and finally 120mg/L protocatechuic acid is obtained.

As can be seen in FIG. 4, the initial concentration of the strain PCA4 was 200 (mg/L), 600 (mg/L), 1000

The protocatechuic acid can be produced under the p-coumaric acid conditions of (mg/L), 1400 (mg/L), 1600 (mg/L) and 2000 (mg/L), wherein the content of the synthesized protocatechuic acid is the highest under the p-coumaric acid condition of 1600mg/L,

can reach 720mg/L.

As shown in FIG. 5, the strain PCA4 was inoculated into YPD medium containing 0.5X lignin hydrolysate and fermented to produce protocatechuic acid at a concentration of up to 400 mg/L.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Sequence listing

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Claims

1. The saccharomyces cerevisiae engineering bacteria are characterized by being knocked outADH6、ADH7 and BDH2The genetic saccharomyces cerevisiae strain is used as a chassis strain and is transferred with exogenous genes4CL、Ech、Fcs、vdhAndPobA(ii) a The starting strain of the chassis strain is a saccharomyces cerevisiae strain BY4742;

among the foreign genes, the gene encoding a polypeptide,4CLthe gene is from parsley; Echthe gene is derived from pseudomonas putida or streptomyces;Fcsthe gene is derived from pseudomonas putida or streptomyces;vdhgenes andpobAthe gene is derived from pseudomonas putida;

the above-mentioned4CLThe sequence of the gene is shown as SEQ ID NO:1 is shown in the specification;

derived from said StreptomycesEchThe accession number of the gene is KC847406.1,Fcsthe accession number of the gene is KC847405.1;

said Pseudomonas putida derivedEchThe accession number of the gene is AAN68962.1，FcsThe accession number of the gene is AAN68960.2,Vdhthe accession number of the gene is AAN68961.1,PobAthe accession number of the gene is AAN69138.1.

2. The engineered saccharomyces cerevisiae strain as claimed in claim 1, wherein the endogenous gene and the exogenous gene are each linked to a promoter at the 5 'end and a terminator at the 3' end.

3. The saccharomyces cerevisiae engineering bacteria of claim 2, wherein the promoter is a saccharomyces cerevisiae endogenous promoter P _TPI1P 、P _FBA1 、P _ENO2 Or P _PGK1 。

4. Use of the engineered strain of saccharomyces cerevisiae of any one of claims 1~3 for the preparation of protocatechuic acid.

5. The method for constructing engineered saccharomyces cerevisiae strain of any of claims 1~3, comprising:

constructing an expression module of a promoter-exogenous gene-terminator, and transforming the expression module and a linearized skeleton vector into a chassis strain.

6. The method of claim 5, wherein the skeletal vector is PRS415 or PRS413.

7. A method for producing protocatechuic acid, which comprises fermenting the yeast engineering bacterium of claim 1~3.

8. The method of claim 7, wherein the fermentation medium comprises coumarin acids or lignin.