CN114525214A

CN114525214A - Construction method and application of engineering probiotics

Info

Publication number: CN114525214A
Application number: CN202210181606.4A
Authority: CN
Inventors: 黄鹤; 吴嘉豪; 王丽娜; 康广博
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2022-02-25
Filing date: 2022-02-25
Publication date: 2022-05-24
Anticipated expiration: 2042-02-25
Also published as: CN114525214B

Abstract

The invention provides an engineering probiotic for excessively synthesizing extracellular butyric acid, a construction method and application thereof in food, health care products or medical products. The probiotics comprises four introduced exogenous genes, wherein the four exogenous genes comprise a 3-hydroxybutyryl-CoA dehydrogenase encoding gene Hbd, an enoyl-CoA hydratase encoding gene Crt, a trans-2-enoyl-CoA reductase encoding gene Ter and butyryl-CoA, namely an acetate CoA transferase encoding gene BCoAT. The probiotic also includes a fifth, sixth, seventh and/or eighth genetic modification. The engineering probiotics of the invention can stably, continuously and efficiently express butyric acid.

Description

Construction method and application of engineering probiotics

Technical Field

The invention belongs to the technical field of genetic engineering and microbial fermentation, and particularly relates to engineering probiotics, a construction method, a fermentation process and application thereof in the fields of medicine, animal husbandry, food, health care or chemical industry.

Background

Butyric acid (also known as n-butyric acid, abbreviated as BA) is a common short-chain fatty acid with a molecular formula of C₄H₈O₂Molecular weight of 88.11, is a volatile colorless oily liquid, and has unpleasant odor and certain irritation. BA is an important member of short chain fatty acid family, and a great deal of research results show that butyric acid is involved in a plurality of important physiological processes, including substance metabolism, cell differentiation, cell autophagy and apoptosis induction, intestinal tissue development promotion, body immunity enhancement and other biological processes, and has important functions on maintaining the normal function of the large intestine and the shape and the function of colon epithelial cells. In clinical applications, butyric acid can relieve and treat a variety of major gastrointestinal disorders, such as colonic inflammation, colon cancer, repair of colonic barrier and coping with oxidative stress, etc., among which, most remarkably, butyric acid has a good anti-Inflammatory effect, and thus it is considered as one of the potential drugs that can relieve and treat major intestinal inflammation, such as Inflammatory Bowel Disease (IBD).

The production method of butyric acid includes chemical synthesis method and fermentation method, but the chemical synthesis method using petroleum as raw material is generally adopted in the current domestic and overseas industrial production, and the method is divided into n-butyl aldehyde oxidation method and propylene carbonyl synthesis method, wherein the former uses n-butyl aldehyde as raw material, oxygen or air as oxidant to carry out oxidation reaction, and reaction liquid is introduced into a rectification system to separate butyric acid; the latter uses propylene and synthesis gas as raw materials, firstly produces butyraldehyde by carbonylation, and then produces butyric acid by air oxidation, and no matter which method, the production investment is large, the operation difficulty of the separation step is large, and because of using petroleum as initial resource, serious environmental pollution is caused. The enormous concern for global warming and the growing demand for butyric acid products have turned the industrial attention to the fermentative production of butyric acid. Compared with a chemical synthesis method, the method for producing butyric acid by a microbial fermentation method has the following advantages: the method has the advantages of mild conditions, simple process, capability of utilizing basic carbon sources, overcoming the defect of excessive byproducts in the chemical synthesis method, less influence on the environment, capability of optimizing fermentation conditions to meet continuous production requirements and the like, and is a production process which has great development prospect and is worthy of development.

Clostridia, strictly anaerobic bacteria, have long been used for microbial fermentative production of butyric acid due to their higher butyric acid production rates. However, the clostridium fermentation model is too complex to operate, and in addition, the progress of the clostridium-based butyrate production process is hindered by the low availability of genetic tools and physiological information, and the bacterial fermentation of butyric acid is also faced with the problems of poor repeatability, unstable yield, difficult industrialization and the like. The probiotics is a kind of active microorganism which is beneficial to a host and changes the flora composition of a certain part of the host in a human body through colonization, and comprises microzyme, probiotic bacillus, clostridium butyricum, lactobacillus, bifidobacterium, actinomycetes and the like, and the yeast, especially Saccharomyces cerevisiae (Saccharomyces cerevisiae) is one of the most important industrial microorganisms, not only has the advantages of mature industrial application technology, strong stress resistance and GRAS (GRAS) microorganism of food safety, but also is one of the most important model organisms of molecular biology and genetic research as the earliest microorganism strain for realizing whole genome sequencing. With the increasing perfection of genetic manipulation means, the basic research of butyrate metabolic pathways and regulatory genes thereof is increasingly deep. Unfortunately, the butyric acid microbial fermentation realized at home and abroad is based on gram-negative bacteria such as clostridium, escherichia coli, bacillus subtilis and the like, and no relevant research on construction of butyric acid producing bacteria by taking probiotics, particularly saccharomyces as a chassis exists, if the butyric acid metabolic pathway is constructed in the probiotics by means of synthetic biology and the probiotic strains are transformed by metabolic engineering to realize excessive production of butyric acid, the cost is greatly reduced, the process is simplified, so that a new way for producing butyric acid by a microbial fermentation process is opened up, and the functions of promoting digestion and absorption of nutrient substances, improving the immunity of organisms and the like of the probiotics are combined, so that the green and clean production of high-value medicines and biochemical products is promoted, and the method has important economic value and social significance.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, provides engineering probiotics which are easy to culture, suitable for large-scale fermentation production and easy to realize industrialization and can produce butyric acid through genetic modification, a recombinant method for constructing the strain and application. In particular, the amount of the solvent to be used,

in a first aspect of the invention there is provided an engineered probiotic comprising modifications of four exogenous genes of butyrate metabolizing enzyme, said four exogenous genes comprising: the first gene encodes 3-hydroxybutyryl-CoA dehydrogenase protein (HBD), the second gene encodes enoyl-CoA hydratase protein (CRT), the third gene encodes trans-2-enoyl-CoA reductase protein (TER), and the fourth gene encodes butyryl-CoA: acetate CoA transferase protein (BCoAT).

The engineering probiotics can synthesize the final product butyric acid through the modification of four exogenous genes. Wherein,

by modifying the first gene, the probiotics can synthesize 3-hydroxybutyryl-CoA by themselves, so that free acetoacetyl-CoA in the probiotics cytoplasm is dehydrogenated to form 3-hydroxybutyryl-CoA, which is the first step of butyric acid synthesis;

by modifying the second gene, the probiotic itself is able to synthesize crotonyl-coa, which under the catalysis of this enzyme can be hydrated on the basis of 3-hydroxybutyryl-coa, which is the second step of butyrate synthesis;

by modifying the third gene, the probiotics can further synthesize butyryl-CoA, and the butyryl-CoA can be continuously reduced to form butyryl-CoA on the basis of crotonyl-CoA under the catalysis of the enzyme, which is the third step of synthesizing butyric acid;

through modifying the fourth gene, the probiotics can synthesize butyryl-CoA, namely, acetic acid CoA transferase, acyl group transfer can be carried out on the basis of the butyryl-CoA under the catalysis of the enzyme so as to form butyric acid, and the final product butyric acid is synthesized.

Preferably, the first gene includes, but is not limited to, the 3-hydroxybutyryl-coa encoding gene HBD derived from Clostridium acetobutylicum or HDAC derived from Faecalibacterium prausnitzii, and more preferably, the first gene is HBD.

The coded protein of the 3-hydroxybutyryl-CoA dehydrogenase gene HBD is a hexamer protein structure formed by two types of dimers, namely a homodimer containing two apo subunits and a heterodimer of apo subunits combined with NAD +, the HBD protein has dependency on NADH/NADPH cofactors, and a sheet-shaped domain with extensibility contained in the protein structure of the HBD protein contains a Rossmann folding topological structure which is crucial to the combination of the cofactors. And the substrate binding site and the cofactor binding site of the acetoacetyl-CoA in the HBD protein structure are oriented vertically to facilitate the transfer of electrons when the acetoacetyl-CoA participates in the reduction reaction.

The amino acid sequence of the HBD protein encoded by HBD can be found, for example, in GenBank: AAA 95971.1.

The nucleotide sequence of HBD can be found, for example, in positions 4088-4936 of GenBank: U17110.1.

The nucleotide sequence of HBD may also be complementary to, or degenerate to, the above sequences, more suitable for codon-optimized sequences for expression in engineered probiotic bacteria.

In a specific embodiment, the nucleotide sequence of HBD: 1) at least 90% identity to SEQ ID NO. 1, preferably at least 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO. 1, and the like; 2) having a deletion, insertion or substitution of one or more nucleotides relative to SEQ ID NO. 1, 3) is capable of hybridizing to SEQ ID NO. 1 under stringent hybridization conditions; 4) 1, a complementary or degenerate sequence as shown in SEQ ID NO; or 5) as shown in SEQ ID NO. 1, the nucleotide sequences have the function of coding HBD protein.

The second gene includes but is not limited to enoyl-coa hydration gene CRT derived from Clostridium acetobutylicum or ECHA derived from Faecalibacterium prausnitzii, and more preferably, the second gene is CRT.

Enoyl-coa hydration gene CRT, which encodes a protein that is a hexamer formed from dimerization of two trimers. The CRT monomer consists of a central domain (NTD) containing the crotonase fold and a Carbon Terminal Domain (CTD) consisting of three a-helices which mediate oligomerization of CRT. In addition, the extended a-helix interacts with the NTD of the adjacent monomer and thus participates in the formation of the substrate binding site. The substrate specificity of the enzyme is determined by the structural features of the helical region of a3 and the residues that form the binding site for enoyl-coa. The close formation of the a3 helix and the two phenylalanine residues, Phe143 and Phe233, contributes to CRT-specific containment of the substrate.

The amino acid sequence of the CRT encoded nucleotide CRT protein can be found, for example, in GenBank: AAA 95967.1.

The nucleotides of CRT can be found, for example, in positions 178-963 of GenBank: U17110.1.

The nucleotide sequence of CRT may also be complementary to, or degenerate to, the above sequences, more suitable for codon-optimized sequences for expression in engineered probiotics.

In a specific embodiment, the nucleotide sequence of the CRT: 1) at least 90% identity to SEQ ID NO. 2, preferably at least 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO. 2, and the like; 2) having a deletion, insertion or substitution of one or more nucleotides relative to SEQ ID NO. 2, 3) is capable of hybridizing to SEQ ID NO. 2 under stringent hybridization conditions; 4) a complementary or degenerate sequence as shown in SEQ ID NO 2; or 5) as shown in SEQ ID NO. 2, the nucleotide sequences have the function of coding the CRT protein.

The third gene is derived from the trans 2-enoyl-CoA reductase gene TER of Treponema denticola or from CCRA of Faecalibacterium praussnintzii, and more preferably the third gene is TER.

The trans 2-enoyl-coa reducing gene TER, which encodes a protein that is typical of SDR enzymes, comprises two domains, a cofactor-binding domain and a substrate-binding domain, with an active site located between the two domains. The cofactor binding domain exhibits a classical Rossmann fold, consisting of a six-stranded parallel beta sheet with five alpha-helices on one side and three alpha-helices on the other side. The substrate binding domain consists of five alpha-helices on one side, two alpha-helices on the other side, one short alpha-helix and one beta-hairpin, and one beta-hairpin covering the top. Certain residues in the active site may play an important role in the TER enzyme catalytic process, Lys 244 plays an important role in cofactor binding and proton transfer; tyr235 participates in the protonation of the substrate thioester carbonyl and the stabilization of the enolate intermediate; tyr225 is primarily involved in the binding and stabilization of the crotyl moiety of the substrate.

The amino acid sequence of TER can be found, for example, in positions 1-397 of GenBank: Q73Q47.1.

The nucleotide sequence of TER can be found, for example, in positions 636109-637302 of GenBank: NC-002967.9.

The nucleotide sequence of the TER can also be a complementary sequence or a degenerate sequence of the sequences, and is more suitable for a codon optimization sequence expressed in the engineering probiotics.

In a specific embodiment, the nucleotide sequence of the TER: 1) at least 90% identity to SEQ ID NO. 3, preferably, at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO. 3, and the like; 2) having a deletion, insertion or substitution of one or more nucleotides relative to SEQ ID NO. 3, 3) is capable of hybridizing to SEQ ID NO. 3 under stringent hybridization conditions; 4) a complementary or degenerate sequence shown in SEQ ID NO 3; or 5) as shown in SEQ ID NO. 3, and the nucleotide sequences have the function of coding TER protein.

The fourth gene is derived from butyryl-CoA of Faecalibacterium praussnitzii, acetate CoA transfer gene BCoAT.

Under the condition of existence of acetic acid, butyryl CoA can transfer acyl to acetic acid under the action of BCoAT so as to form butyric acid and form byproduct acetyl CoA. The coupling of butyrate metabolic synthesis to acetate is an important characteristic of butyrate high-producing strains, and the coupling can reduce the concentration of acetate and simultaneously can increase the accumulation of butyrate and acetyl coenzyme A.

The amino acid sequence of the BCoAT-encoded protein can be found, for example, in GenBank: QIA 42843.1.

The BCoAT nucleotide sequence can be found, for example, in GenBank: CP048437.1 at positions 1460292-1461638.

The nucleotide sequence of BCoAT may also be complementary to, or degenerate to, the above sequences, more suitable for codon-optimized sequences for expression in engineered probiotics.

In a specific embodiment, the nucleotide sequence of BCoAT: 1) at least 90% identity to SEQ ID NO. 4, preferably, at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO. 4, and the like; (ii) a

2) Having a deletion, insertion or substitution of one or more nucleotides relative to SEQ ID NO. 4, 3) is capable of hybridizing to SEQ ID NO. 4 under stringent hybridization conditions; 4) a complementary or degenerate sequence as shown in SEQ ID NO 4; or 5) as shown in SEQ ID NO. 4, the nucleotide sequences have the function of coding BCoAT protein.

Compared with the engineering probiotics without gene modification, the modified engineering probiotics obtain the capacity of producing butyric acid by modifying the four exogenous genes of the butyric acid metabolic enzyme, and the primary yield of the butyric acid can reach at least 180mg/L, such as 193 mg/L.

Preferably, the four genes are modified to increase or overexpress the activity of the encoded enzyme, and more preferably, the modified genes to increase or overexpress the activity of the encoded enzyme include point mutations, linkage to a strong promoter, linkage to an enhancer, increase in copy number, or fusion co-expression. Further preferably, the genetic modification comprises linking a strong promoter, and more preferably, the strong promoter comprises a PGK1 promoter, a TPI1 promoter, a TEF1 promoter, a TDH3 promoter and the like. Preferably, the engineered probiotic bacteria further comprise a fifth genetic modification to increase the positive regulation of the synthesis of the precursor acetoacetyl-coa in the butyrate synthesis pathway.

More preferably, said fifth genetic modification modifies the endogenous gene Acetyl-coenzyme A C-acetyltransferase (Acetyl-CoA C-acetyltransferase) ERG10, and even more preferably, said fifth genetic modification increases or overexpresses the activity of the enzyme encoded by the modified gene. The acetyl-CoA is polymerized to form acetoacetyl-CoA under the action of the protein ERG10, so that the substrate accumulation amount of the acetoacetyl-CoA is enhanced, and the generation amount of butyric acid is increased.

ERG10 transfers acetyl groups from one acetyl-coa molecule to another, forming acetoacetyl-coa cytosolic enzymes.

The amino acid sequence of the protein encoded by ERG10 can be found, for example, in GenBank: KAF 1901504.1.

The nucleotide sequence of ERG10 can be found, for example, in GenBank: JAAEAL010000015.1 at positions 498528-499724.

The nucleotide sequence of ERG10 may also be a complement, or degenerate, of the above sequences, more suitable for codon-optimized sequences for expression in engineered probiotics.

Preferably, the fifth gene modification includes, but is not limited to, point mutation, linkage to a strong promoter, linkage to an enhancer, increased copy number or fusion co-expression. More preferably, the genetic modification comprises linking a strong promoter to increase the copy number, and more preferably, the strong promoter comprises PGK1 promoter, TPI1 promoter, TEF1 promoter, TDH3 promoter and the like.

Compared with the engineering probiotics modified by four genes, the modified engineering probiotics are enhanced in butyric acid synthesis capacity, and the butyric acid yield is increased by at least 25%, such as 25%, 28%, 30%, 32%, 35%, 40% and even higher by the five gene modifications.

Preferably, the engineered probiotic bacteria further comprise a sixth genetic modification to increase the positive regulation of the synthesis of the precursor acetoacetyl-coa in the butyrate synthesis pathway.

More preferably, the sixth genetic modification employs the endogenous genes malonyl-coa: ACP transferase (malonyl-CoA: ACP transferase) MCT1, malonyl-CoA forms acetoacetyl-CoA under the action of MCT1 corresponding to the catalytic enzyme. Further preferably, the sixth genetic modification results in an increased activity or overexpression of the enzyme encoded by the modified gene. Further strengthening the accumulation of acetoacetyl-CoA and increasing the production of butyrate on the basis of the fifth gene modification.

The amino acid sequence of the protein encoded by MCT1 can be found, for example, in GenBank: KAF 1902377.1.

The nucleotide sequence of MCT1 can be found, for example, in GenBank: JAAEAL010000014.1 at positions 762615-763697.

The nucleotide sequence of MCT1 may also be a complement, or degenerate, of the above sequences, more suitable for codon-optimized sequences for expression in engineered probiotics.

Further preferably, the sixth gene is modified by a method including, but not limited to, point mutation, linkage to a strong promoter, linkage to an enhancer, increase in copy number or fusion co-expression. More preferably, the genetic modification comprises linking a strong promoter to increase the copy number, and more preferably, the strong promoter comprises PGK1 promoter, TPI1 promoter, TEF1 promoter, TDH3 promoter and the like.

Compared with the four or five genetically modified engineering probiotics, the modified engineering probiotics are further enhanced in butyric acid synthesis capacity, and the butyric acid yield is further increased by at least 25%, such as 25%, 28%, 31%, 35%, 40%, 45%, or even higher by the six genetic modifications.

Preferably, the above engineered probiotic bacteria further comprise a seventh genetic modification to increase acetoacetyl-coa by increasing the regulation of acetyl-coa, for example, to decrease the amount of acetyl-coa split, thereby further increasing butyrate production.

More preferably, the seventh gene is Malate synthase (MLS1), and the seventh gene is modified such that the activity of the enzyme encoded by the modified gene is decreased or the expression thereof is suppressed.

The acetyl-CoA can form malic acid under the action of MLS1, the splitting flow of the acetyl-CoA is reduced due to the reduction of MLS1 activity or the inhibition of expression, the accumulation amount of the acetyl-CoA is increased, and the yield of butyric acid is enhanced.

MLS1 is malate synthase gene participating in glyoxylate cycle, and glyoxylate and acetyl-CoA can form malate under the action of MLS 1.

The amino acid sequence of the protein encoded by MLS1 can be found, for example, in GenBank: KAF 1903065.1.

The nucleotide sequence of MLS1 can be found, for example, in GenBank: JAAEAL010000013.1 at positions 387832-389496.

Further preferably, the seventh gene modification means includes, but is not limited to, point mutation, deletion, insertion, antisense polynucleotides, siRNA, microRNA, CRISPR, and more preferably, the modification means includes point mutation, deletion or insertion of one or more nucleotides, and the point mutation is, for example, conversion of a single nucleotide (purine to purine or pyrimidine to pyrimidine) or transversion (purine to pyrimidine or pyrimidine to purine). Mutations in nucleotides can result in the substitution of one or more conserved or non-conserved amino acids in the polypeptide that it expresses, which may result in a change in the conformation of the polypeptide, which may also lose part or all of its function, which may result in a frame-shift mutation that results in the entire polypeptide chain encoding a completely different polypeptide from that point, which may also lead to the premature formation of a stop codon that causes the polypeptide chain to be defective or even silenced.

The nucleotide sequence for deletion or insertion may be obtained by PCR or chemical synthesis, or may be obtained by cell replication and amplification. The gene modification to completely inhibit the activity or expression of the gene enzyme can also be realized by providing or expressing antisense oligonucleotides, siRNA, microRNA or other nucleotides which can prevent the translation of mRNA of the gene to be modified into protein. Recently developed transcription activator-like effector nucleases (TALENs) and crisp technology, Clustered Regularly Interspaced Short Palindromic Repeats, can also be used to inactivate genes.

Reference herein to a genetic modification that results in a reduction in the enzymatic activity of a gene or suppression of expression is to be taken to mean that the activity of such an enzyme/polypeptide is reduced by at least 95% (e.g. at least 96%, 97%, 98% 99%, or 100%) compared to the activity of the non-genetically modified probiotic or wild-type probiotic.

In a specific embodiment, the nucleotide sequence of MLS1 is partially or fully deleted, the deletion being at least 8bp, such as 8, 10, 15, 20, 30, 50, 80, 100 or more. Preferably, the nucleotide deletion portion of MLS1 is located in a functional region of the MLS1 protein, e.g., corresponding to the deletion at position 170-200 of the MLS 1-encoding protein, more preferably, corresponding to the deletion at position 175-190 of the MLS 1-encoding protein, even more preferably, corresponding to the deletion at position 179-182 of the MLS 1-encoding protein.

Compared with the four, five or six genetically modified engineering probiotics, the modified engineering probiotics with the seventh genetic modification have the advantages that the butyric acid synthesis capacity is further enhanced, and the yield is improved by at least 15%, such as 15%, 18%, 20%, 22%, 25%, 28%, 30% or even higher.

Preferably, the engineering probiotics further comprise an eighth gene modification, and the accumulation amount of acetyl coenzyme A is further increased and the yield of butyric acid is increased by increasing the regulation and control of acetyl coenzyme A.

More preferably, the eighth gene is Acetyl-CoA Synthetase ACS1, and the eighth gene is modified to increase or over-express the activity of the enzyme encoded by the modified gene.

The acetate forms acetyl coenzyme A under the action of ACS1 corresponding to catalytic enzyme, and the substrate accumulation of acetyl coenzyme A is enhanced due to the improvement or overexpression of ACS1 activity, so that the yield of butyrate is further improved.

Further preferably, the modification of the eighth gene includes, but is not limited to, point mutation, linkage to a strong promoter, linkage to an enhancer, increase in copy number or fusion co-expression. More preferably, the genetic modification comprises linking a strong promoter to increase copy number.

The amino acid Sequence of the protein encoded by ACS1 can be found, for example, in NCBI Reference Sequence: NP-013254.1.

The nucleotide Sequence of ACS1 can be found, for example, in NCBI Reference Sequence: NM-001182040.1.

The nucleotide sequence of ACS1 may also be a complement, or degenerate, sequence to the wild-type sequence described above, more suitable for codon-optimized sequences for expression in engineered probiotics.

Through the eighth gene modification, compared with the four, five, six or seven gene-modified engineering probiotics, the butyric acid synthesis capacity of the modified engineering probiotics is further enhanced, and the yield is improved by at least 10%, such as 10%, 12%, 14%, 16%, 18 |%, 20% or even higher.

Such modifications of the gene that result in increased or over-expression of the activity of the encoded enzyme include, but are not limited to, linking a strong promoter, increasing copy number. For example, linking a nucleotide sequence to an expressible strong promoter or enhancer by genetic modification; altering the promoter or other regulatory factor of the nucleotide sequence to increase its level of expression (e.g., increasing the binding strength of the promoter sequence to the transcription initiation factor); the expression level of the corresponding endogenous gene is improved by inserting a yeast endogenous nucleotide sequence expression frame capable of high expression into other sites through gene modification.

Preferably, the strong promoter includes PGK1 promoter, TPI1 promoter, TEF1 promoter, TDH3 promoter, and the like.

More preferably, the Hbd gene is linked to the PGK1 promoter;

the Crt gene is connected with TPI1 promoter;

the Ter gene is connected with a PGK1 promoter;

the BCoAT gene is connected with a TPI1 promoter;

the ERG10 gene is connected with a TEF1 promoter;

the MCT1 gene is connected with a TDH3 promoter;

the ACS1 gene was linked to the TEF1 promoter.

As used herein, a genetic modification that results in an increase or overexpression of the enzymatic activity of a gene refers to an increase of at least 20% (e.g., at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more) in the activity or efficiency of action on a substrate of an enzyme/polypeptide encoded by a genetically modified nucleotide sequence as compared to the wild type.

In addition to the necessity of simultaneously expressing heterologously the combination of the four butyrate-critical synthetic genes HBD, CRT, TER, BCoAT in the engineered probiotic, the remaining fifth to eighth genes may be modified in no way, singly or in any combination, except that the expression of the first four enzyme genes is modified in such a way that the corresponding enzyme activity is increased or overexpressed. The fifth, sixth, seventh or eighth genes are only named after each other for distinguishing and do not indicate chronological order, for example, the seventh gene may be modified after the first to fourth genes are modified, and then the fifth gene may be modified, or for example, the eighth gene may be modified after the first to fourth genes are modified, and then the fifth gene may be modified, and so on. Preferably, the engineered probiotic bacteria comprise one or more gene modifications of a fifth gene to an eighth gene, more preferably, the engineered probiotic bacteria comprise one or more gene modifications of a fifth gene, a seventh gene and an eighth gene, and even more preferably, the engineered probiotic bacteria comprise a modification of a sixth gene.

On the other hand, the generation of butyrate requires that acetyl-CoA is formed after 5 reactions of transfer, dehydrogenation, hydration and reduction are carried out in sequence under the catalysis of 5 key enzymes, so that the accumulation amount of acetyl-CoA can influence the expression activity of the 5 enzymes, and further can influence the yield of butyrate. Acetic acid in the engineering probiotics is used as a competitive metabolite of butyric acid, and excessive synthesis can cause the bypass metabolic shunt of acetyl coenzyme A to be increased, thereby influencing the yield of butyric acid. The fourth gene enzyme described by the invention can transfer the acyl carrier of the pro-substrate butyryl-CoA to acetic acid, form the final product butyric acid and simultaneously form acetyl-CoA by using the acetic acid, thereby reducing the content of the acetic acid and increasing the accumulation amount of the acetyl-CoA. In order to further improve the yield of acetyl coenzyme A, the fifth gene enzyme described by the invention can reduce the malic acid synthesis rate, reduce the bypass metabolic flux of acetyl coenzyme A and further improve the accumulation amount; the sixth gene enzyme can strengthen the metabolism amount of acetic acid to acetyl coenzyme A, reduce the yield of competitive metabolite acetic acid and further strengthen the accumulation amount of acetyl coenzyme A, thereby further strengthening the yield of butyric acid.

Further, the modification of the gene comprises the use of a selectable marker gene. Preferably, the selectable marker genes include, but are not limited to, URA3, LEU2, HIS3, TRP 1. More preferably, the URA3 selection marker gene is used. Even more preferably, the gene is modified in such a way that the selectable marker gene is recycled so that the selectable marker gene is not retained.

Compared with yeast without gene modification, the engineering probiotics described by the invention obtains a certain level of butyric acid synthesis capacity, and the synthesis level is obviously enhanced. In a most preferred embodiment, the engineered probiotic bacteria described herein have butyric acid production of 465 mg/L.

Preferably, the engineered probiotics include yeast, probiotic bacillus, clostridium butyricum, lactobacillus, bifidobacterium, actinomycetes, and the like, and more preferably, the yeast includes any one of Saccharomyces cerevisiae (Saccharomyces cerevisiae), Saccharomyces pastorianus (Saccharomyces pastorianus), pichia stipitis (Pichiastipitis), Saccharomyces Bayanus and Candida huyata (Candida shehatae). More preferably, the yeast is Saccharomyces cerevisiae (Saccharomyces cerevisiae).

In a second aspect of the present invention, there is provided a method for constructing the above engineering probiotics, the method comprising:

1) introducing modification of four exogenous genes for metabolism of butyric acid into engineering probiotics, so that the engineering probiotics sequentially react and synthesize 3-hydroxybutyryl coenzyme A, crotonyl coenzyme A, butyryl coenzyme A and butyric acid on the basis of taking acetoacetyl coenzyme A as a substrate, and complete 4-step reaction of synthesis of butyric acid, wherein the four exogenous genes comprise: the first gene encodes 3-hydroxybutyryl-CoA dehydrogenase protein (HBD), the second gene encodes enoyl-CoA hydratase protein (CRT), the third gene encodes trans-2-enoyl-CoA reductase protein (TER), and the fourth gene encodes butyryl-CoA: acetate CoA transferase protein (BCoAT).

Preferably, the construction method further comprises 2) introducing a fifth genetic modification, a sixth genetic modification, a seventh genetic modification, and/or an eighth genetic modification to the engineered probiotic bacteria, wherein the fifth and sixth genetic modifications increase the positive regulation of the synthesis of the precursor acetoacetyl-coa in the butyrate synthesis pathway. The seventh gene modification reduces the shunting amount of acetyl coenzyme A, increases the accumulation amount of acetyl coenzyme A, regulates the accumulation of acetyl coenzyme A and leads the synthesis and secretion of butyric acid to be increased. And the eighth gene modification is to increase the accumulation of acetyl coenzyme A and strengthen the butyric acid synthesis capacity of the engineering probiotics.

More preferably, said fifth genetic modification modifies the endogenous gene Acetyl-coenzyme A C-acetyltransferase (Acetyl-CoA C-acetyltransferase) ERG 10; a sixth genetic modification is directed to the endogenous gene malonyl-coa: ACP transferase (malonyl-CoA: ACP transferase) MCT 1; the seventh gene modification modifies Malate synthase (Malate synthase) MLS 1; the eighth gene modification modifies the endogenous Acetyl-CoA Synthetase (Acetyl CoA Synthetase) ACS 1.

More preferably, the first to sixth and eighth gene modifications increase or overexpress the activity of the enzyme encoded by the modified gene, and further preferably, the gene modifications that increase or overexpress the activity of the encoded enzyme include point mutations, strong promoters linked, enhancers linked, increased copy number or fusion co-expression.

Further preferably, the strong promoter includes PGK1 promoter, TPI1 promoter, TEF1 promoter, TDH3 promoter and the like.

More preferably, the seventh gene is modified by reducing the activity of the enzyme or inhibiting the expression of the enzyme encoded by the modified gene, and the seventh gene is modified by a method including, but not limited to, point mutation, deletion, insertion, antisense polynucleotides, siRNA, microRNA, CRISPR, and more preferably, the modified gene comprises point mutation, deletion or insertion of one or more nucleotides, and the point mutation is a transition of a single nucleotide (purine to purine or pyrimidine to pyrimidine) or a transversion (purine to pyrimidine or pyrimidine to purine). Mutations in nucleotides can result in the substitution of one or more conserved or non-conserved amino acids in the polypeptide that it expresses, which may result in a change in the conformation of the polypeptide, which may also lose part or all of its function, which may result in a frame-shift mutation that results in the entire polypeptide chain encoding a completely different polypeptide from that point, which may also lead to the premature formation of a stop codon that causes the polypeptide chain to be defective or even silenced.

The nucleotide sequence for deletion or insertion may be obtained by PCR or chemical synthesis, or may be obtained by cell replication and amplification. The gene modification to completely inhibit the enzymatic activity or expression of the gene can also be achieved by providing or expressing antisense oligonucleotides polynucleotides, siRNA, microRNA or other nucleotides that prevent translation of mRNA of the gene to be modified into protein. Recently developed transcription activator-like effector nucleases (TALENs) and crisp technology, Clustered Regularly Interspaced Short Palindromic Repeats, can also be used to inactivate genes.

After said modification the nucleotide sequence of the endogenous seventh gene is partially or completely deleted, said deletion being at least 8bp, such as 8, 10, 15, 20, 30, 50, 80, 100 or even more. Preferably, the nucleotide deletion portion of MLS1 is located in a functional region of MLS1 protein, e.g., corresponding to the deletion at position 170-182 of MLS1 protein, more preferably, corresponding to the deletion at position 175-190 of MLS1 protein, and still more preferably, corresponding to the deletion at position 179-182 of MLS1 protein.

Further preferably, the step 1) includes: (1-1) constructing a vector comprising the first and/or second gene; (1-2) introducing the vector of the step (1-1) into engineering probiotics to obtain engineering probiotics containing a first gene and/or a second gene; (1-3) constructing a vector comprising the third and/or fourth species; (1-4) introducing the vector in the step (1-3) into the yeast strain in the step (1-2) to obtain the engineering probiotics containing the first, second, third and fourth genes.

The step 2) comprises the following steps: (2-1) constructing a vector comprising the fifth, sixth, seventh and/or eighth gene; (2-2) introducing the vector obtained in the step (2-1) into the engineering probiotics obtained in the step 1), and obtaining the vector containing various genetic modifications.

Preferably, the vector comprises regulatory elements, and further preferably, the regulatory elements include promoters, terminators, and the like.

More preferably, the vector comprises:

the Hbd gene is linked to the PGK1 promoter (first gene);

the Crt gene is linked with the TPI1 promoter (second gene);

the Ter gene is connected with a PGK1 promoter (a third gene);

the BCoAT gene was linked to the TPI1 promoter (fourth gene);

the ERG10 gene is connected with a TEF1 promoter (a fifth gene);

the MCT1 gene is connected with a TDH3 promoter (a sixth gene); and/or the presence of a gas in the gas,

the ACS1 gene was linked to the TEF1 promoter (eighth gene).

Further, the vector also comprises a left homology arm sequence and a right homology arm sequence. Preferably, the introducing comprises gene editing using CRISPR/Cas 9. More preferably, the gRNA is introduced together with the vector.

In a specific embodiment, the step 1) comprises: (1-1) construction of vector V comprising first and second genes^H+CThe vector sequentially comprises a H1 left homologous arm, a PGK1 promoter, a Hbd gene, a PGK1 terminator, a TPI1 promoter, a Crt gene, an Adh1 terminator and an H2 right homologous arm; (1-2) introducing the vector obtained in the step (1-1) into engineering probiotics to obtain engineering probiotics containing a first gene and a second gene; (1-3) construction of vector V comprising third and fourth species^T+BThe vector sequentially comprises a left H1 homology arm, a PGK1 promoter, a Ter gene, a PGK1 terminator, a TPI1 promoter, a BCoAT gene, an Adh1 terminator and a right H2 homology arm; (1-4) step (ii)And (1) introducing the vector of (1-3) into the yeast strain of the step (1-2) to obtain the engineering probiotics containing the first, second, third and fourth genes.

More preferably, the vector V comprising the fifth gene^ESequentially comprises a left homologous arm of H1, a TEF1 promoter, an ERG10 gene, a TEF1 terminator and a right homologous arm of H2;

the vector V comprising the sixth gene^MSequentially comprises a left homologous arm of H1, a TDH3 promoter, an MCT1 gene, a TDH3 terminator and a right homologous arm of H2;

the vector V comprising the fifth and sixth genes^E+MSequentially comprises a left homologous arm of H1, a TEF1 promoter, an ERG10 gene, a TEF1 terminator, a TDH3 promoter, an MCT1 gene, a TDH3 terminator and a right homologous arm of H2;

the vector V comprising the seventh gene^MLSequentially comprises a left homology arm of H1, a donor fragment with partial or total deletion and a right homology arm of H2;

the vector V comprising the eighth gene^ASequentially comprises a left homologous arm of H1, a TEF1 promoter, an ACS1 gene, a TEF1 terminator and a right homologous arm of H2.

In addition to the necessity of simultaneously expressing heterologously the combination of the four butyrate-critical synthetic genes HBD, CRT, TER, BCoAT in the engineered probiotic, the remaining fifth to eighth genes may be modified in any combination, not sequentially, regardless of the manner in which the expression of the first four enzyme genes is modified to increase or overexpress the corresponding enzyme activities. The fifth, sixth, seventh or eighth genes are only named after each other for distinguishing and do not indicate chronological order, for example, the seventh gene may be modified after the first to fourth genes are modified, and then the fifth gene may be modified, or for example, the eighth gene may be modified after the first to fourth genes are modified, and then the fifth gene may be modified, and so on.

Further preferably, the nucleotide sequence (e.g., expression vector) may be introduced into the probiotic cell or other host cell by a variety of methods. These methods include, but are not limited to, electroporation, calcium phosphate precipitation, heat shock, lipofection, microinjection, lithium chloride, lithium acetate, and virus-mediated gene transfer.

In addition to probiotic cells, the host cell may be any cell that can be used for standard molecular biology procedures and produces nucleotides and polypeptides. Including but not limited to bacterial cells (e.g., e.coli), insect cells, plant cells, and mammalian cells (e.g., CHO or COS cells). Probiotic cells include engineered probiotic cells described herein. The host cell referred to herein includes not only the parent cell to which nucleotide transduction is performed but also daughter cells thereof.

In the genetic modification process of the present invention, the selection marker gene for screening transferred probiotic cells includes, but is not limited to, any auxotrophic gene available for probiotic bacteria, such as URA3, LEU2, HIS3, TRP1, LYS 2. The URA3 gene is preferably used repeatedly and cyclically, so that it can be modified as many genes as possible while avoiding any interference with the expression of auxotrophic genes themselves. Still more preferably, the selection marker gene such as URA3 is recycled without remaining in the genetically modified strain.

Although the strategy applied by the present invention is directed to the genes and polypeptides of the s. Suitable strains include, in addition to the s.cerevisiae strain, Saccharomyces pastorianus (Pichia stipitis), Pichia stipitis, Saccharomyces Bayanus and Candida huhatensis (Candida shehatae). Likewise, these strategies are also suitable for other probiotics, such as probiotic bacillus, clostridium butyricum, lactobacillus, bifidobacterium, actinomycetes, etc., and other exogenous genes for synthesizing butyric acid, including one or more of the first to eighth genes, may be introduced according to the difference of endogenous genes in these probiotics, and codons may be optimized according to the difference of probiotics. Pathway or gene names may differ slightly in these strains, but the same strategies and techniques can be applied to modify the corresponding pathways and homologous genes. Genetically engineered probiotics described herein are well known to those of skill in the art.

In a third aspect of the invention, there is provided a method for producing butyric acid using the above engineered probiotic, said method comprising culturing the above engineered probiotic in a culture medium.

Preferably, the culture medium in the method comprises a carbon source, a nitrogen source and trace elements.

More preferably, in the method, the culture medium comprises glucose with a concentration of 16-25g/L, yeast extract with a concentration of 8-12g/L and peptone with a concentration of 15-25 g/L.

In a fourth aspect of the invention, a fermentation product produced by fermentation of the engineering probiotics is provided.

Preferably, the fermentation product comprises the engineered probiotic bacteria and secreted butyric acid.

More preferably, the butyric acid content is at least 180mg/L, such as 190, 200, 230, 250, 280, 300, 320, 350, 380, 400, 450, 460mg/L or more, or even higher.

In a fifth aspect of the invention there is provided the use of any one of the engineered probiotics or fermentation products described above in the production of butyric acid.

In a sixth aspect of the present invention, there is provided an application of any one of the above engineered probiotics or fermentation products in the fields of medicine, animal husbandry, food, health products or chemical industry.

Preferably, the application is as a feed or feed additive for animal husbandry.

The seventh aspect of the invention provides an application of any one of the above engineering probiotics or fermentation products in the preparation of products in the fields of medicines, foods, health products or chemical industry and the like.

In an eighth aspect, the invention provides a method for preparing a product in the fields of medicine, animal husbandry, food, health care products or chemical industry, the method comprising preparing the product by using any one of the above engineering probiotics or any one of the above fermentation products.

Preferably, the method comprises further processing the engineered probiotic bacteria or the fermentation product into any other dosage form, more preferably, the dosage form comprises an edible dosage form, further preferably, the dosage form is in the form of a powder, granule, tablet, capsule, or liquid. More preferably, the powder is prepared by a low temperature drying or spray drying method.

In a ninth aspect, the invention provides a formulation comprising any of the engineered probiotics described above or any of the fermentation products described above.

Preferably, the formulation includes any dosage form, more preferably, the dosage form includes an edible dosage form, and further preferably, the dosage form is a powder, granule, tablet, capsule, or liquid form. More preferably, the powder is prepared by a low temperature drying or spray drying method.

Unless otherwise defined, all terms of art and concepts related to herein are well known to those skilled in the art. Although a variety of similar or equivalent methods may be used to construct and determine the engineered probiotics involved in the present invention, one suitable method and material is described below. These materials, methods, and examples are illustrative only and are not intended to limit the present invention in any way. All documents, patents, and other references mentioned herein are incorporated by reference in their entirety. The methods and materials involved in the present invention are set forth below in conjunction with the detailed description and figures.

Advantages and benefits of the invention

1. The method introduces a butyric acid production way into the engineering probiotics for the first time, and the obtained recombinant engineering probiotics have the properties of low nutritional requirement, easy culture, suitability for large-scale fermentation production of butyric acid and easy realization of industrialization;

2. related gene modification is carried out by means of genetic engineering, when 4 exogenous key butyric acid synthetic genes are introduced, the engineering probiotics are endowed with the capacity of preliminarily synthesizing butyric acid, the metabolic flux of a butyric acid precursor acetoacetyl-CoA is accelerated, the bypass metabolic shunt flux of key substance acetoacetyl-CoA of the acetoacetyl-CoA precursor is reduced, the metabolic flux of acetic acid to acetyl-CoA is enhanced, the intracellular and extracellular transportation and accumulation of butyric acid and precursor substance acetoacetyl-CoA of the butyric acid are improved, the obtained recombinant engineering probiotics can stably, continuously and efficiently produce butyric acid, and the yield can reach 400 mg/L.

3. The invention optimizes the nucleotide sequence of each gene, so that the gene is more suitable for being expressed in engineering probiotics, has higher protein activity, and further improves the yield of the final product butyric acid. After four genes are modified, the butyric acid yield can reach 193mg/L, and after the fifth gene to the eighth gene are modified, the butyric acid yield can reach 465 mg/L.

4. The invention overcomes the defects of complex process, expensive raw materials, more byproducts and serious environmental pollution of the traditional chemical synthesis method, has the advantages of simple process operation, low raw material cost, safety, environmental protection and industrialized production, opens up a new way for producing butyric acid by microbial fermentation, combines the functions of promoting the digestion and absorption of other nutrient substances, improving the immunity of organisms and the like of probiotics, promotes the green and clean production of high-value medicines and biochemical products, can be widely applied to the fields of medicines, foods or chemical industry, and has important economic value and social significance.

Drawings

FIG. 1 is a vector construction diagram, the upper diagram is a single-gene vector construction diagram, and the lower diagram is a double-gene vector construction diagram.

Fig. 2 is a Cas9 plasmid construction scheme.

FIG. 3 is a schematic diagram of gRNA plasmid construction.

Detailed Description

The technical solution of the present invention is further described with reference to the following specific examples. It will be understood that the specific embodiments described herein are shown by way of example and not as limitations of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The main features of the invention may be used in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.

Several media involved in the examples are as follows:

(1)LB(Luria Bertani)

liquid: yeast powder (w/v): 0.5%, tryptone (w/v): 1%, NaCl (w/v): 1%, adjusting the pH of the medium to 7.5, and sterilizing at 121 deg.C for 15 min.

Solid: agar powder was added to the liquid LB medium to a final concentration of 1.5% (w/v), sterilized, and poured out after slight cooling.

LBA medium: ampicillin was added to the sterilized LB liquid or solid medium at a concentration of 100. mu.g/mL as conventionally used.

(2) YPD medium

Liquid: yeast extract (w/v): 1%, peptone (w/v): 2%, sterilizing at 121 deg.C for 15min, cooling the culture medium to no scald, and adding 40% (w/v) glucose mother liquor (requiring separate sterilization: 115 deg.C, 25min) to make the final glucose concentration of the culture medium 2% (w/v).

Solid: adding agar powder to YPD liquid culture medium to make its final concentration 1.5% (w/v), sterilizing, and cooling.

(3) Complete ultimate omitting Component Medium (CM)

YNB(w/v)：0.67％，

Dropout powder (w/v): 0.083%, which contains the following final concentrations of components (mg/L):

threonine 150, serine 150, valine 150, glutamic acid 10, aspartic acid 100, phenylalanine 50, lysine 30, tyrosine 30, isoleucine 30, arginine 20, methionine 20;

other nutritional ingredients (mg/L):

histidine 100, leucine 100, tryptophan 100, uracil 50, adenine 50;

the pH range of the liquid culture medium is 5.6-5.8; the pH range of the solid medium is about 6.5.

(4) CMG medium

Liquid culture medium: adding a glucose mother liquor with the final concentration of 2% into a culture medium of the CM to obtain a CMG culture medium;

solid medium: agar powder (w/v) was added to the liquid CMG medium at a final concentration of 1.5%.

CMG + HyB 300 solid medium: and adding a proper amount of hygromycin B into the CMG liquid or solid culture medium to prepare an antibiotic screening plate.

In addition, the deletion of partial amino acids on the basis of the CM medium can be prepared into an auxotrophic screening medium/plate.

(5) 5-Fluorotic acid Medium (5' -FOA)

I. Solution I: 50mL of the following 5' -FOA solutions were prepared, which included: YNB AA: 0.7g, dropout powder: 0.085g, 5' -FOA: 0.1g, uracil: 10mg, adenine: 5mg, leucine: 20mg, histidine: 15mg, tryptophan: 10mg, glucose: 2g of the total weight of the mixture; dissolving at 45 deg.C, filtering, sterilizing, and storing.

II. Solution II: 50mL of a 3% final agar powder solution was prepared, sterilized at 121 ℃ for 15min, and then cooled to 45 ℃. And carefully mixing the solution I and the solution II uniformly to avoid generating bubbles, quickly pouring the mixture into a sterile culture dish, and cooling and forming the mixture for later use.

The primer sequences referred to in the examples are shown in Table 1

Table 1: primer sequences

Example 1: construction of yeast strains inserted with foreign genes of Hbd and Crt

And (3) simultaneously inserting Hbd and Crt genes into a yeast chromosome by using a CRISPR/Cas9 system. Firstly, a vector plasmid YCplac33-Cas9 of Cas9 protein is transformed into yeast competent cells by a lithium acetate method, and then a positive yeast transformant with a Cas9 plasmid is screened by using URA3 as a screening marker. Then pRS42H-gHbd/Crt for guiding Cas9 protein to cut at a specific site and a donor fragment for repairing a DNA chain are constructed, a gRNA expression plasmid and the donor fragment are converted into yeast competent cells, PCR verification is carried out on positive transformants screened BY using URA3 and hygromycin as double screening markers, and a yeast strain BY4741-Hbd-Crt with homologous recombination is screened out

The primers used for construction are shown in Table 1, and schematic diagrams are shown in FIGS. 1-3.

Firstly, the method comprises the following steps: construction of pRS42H-gHbd/Crt plasmid

In the study, pRS42H-gRNA-NotI plasmid linearized by NotI digestion is used as a template, sequences shown in IPCR-Chr IV site3-UP and IPCR-Chr IV site3-DW in the table 1 are used as upstream and downstream primers to carry out reverse PCR on the linearized template, DNA polymerase produced by Nozak company is annealed at 54 ℃ for 15 seconds and extended at 72 ℃ for 4min for 30 cycles to obtain a 6600bp PCR fragment. And (3) carrying out self-ligation reaction on the PCR fragment by using a seamless recombination kit, so that homologous sequences at two ends of the fragment are connected to form pRS42H-gRNA-ORF plasmid (ORF is a gene of a target site) capable of specifically recognizing the corresponding site. 10V is transformed to be competent in large intestine, and after positive transformants grow on a screening plate (LB + AMP100), transformant colonies are expanded and cultured by an LBA liquid medium and are shaken overnight at 37 ℃ and 230rpm, and plasmids are extracted for sequencing verification. Plasmid pRS42H-gHbd/Crt was obtained. The structure of the gRNA sequence is shown in SEQ ID NO: 77.

II, secondly: Hbd/Crt donor fragment construction

The nucleotide sequence of Hbd/Crt derived from Clostridium acetobutylicum is searched at NCBI, and the related gene sequence is subjected to gene synthesis by Jinzhi organism company and codon bias optimization. Performing PCR reaction by using Chr IV site3H 1-UP and Chr IV site3H 1-DW in the table 1 and using a yeast chromosome as a template to obtain a left homologous arm fragment of H1, wherein the structure of the left homologous arm fragment is shown as 544265-544486 of Genbank BK 006938.2; PCR reaction is carried out by using Chr IV site3 pPGK1-UP and Chr IV site3 pPGK1-DW in the table 1 and yeast chromosome as a template to obtain a PGK1 promoter fragment, and the structure of the PGK1 promoter fragment is as Genbank: 137146-137745 of BK 006937.2; by using Hbd-UP and Hbd-DW in the table 1, a PCR reaction is carried out by taking the synthetic gene as a template to obtain an Hbd target gene fragment, and the structure of the Hbd target gene fragment is shown as SEQ ID NO. 1; PCR reaction is carried out by using Chr IV site3 tPGK1-UP, Chr IV site3 tPGK1-DW in the table 1 as primers and yeast chromosome as a template to obtain a PGK1 terminator fragment, wherein the structure of the fragment is as Genbank: as shown at 138997-139440 of BK 006937.2.

The TPI1 promoter fragment is obtained by PCR reaction with Chr IV site3 pTP1-UP, Chr IV site3 pTP1-DW in the table 1 as primers and yeast chromosome as a template, and has the structure as Genbank: the structure of the gene fragment is shown as SEQ ID NO 2, and the PCR reaction is carried out by using Chr IV site3 tAdh1-UP, Chr IV site3 tAdh1-DW in the table 1 as a primer and yeast chromosome as a template to obtain an Adh1 terminator fragment, wherein the structure is shown as Genbank: the right homologous arm fragment of H2 is obtained by PCR reaction using Chr IV site3H2-UP, Chr IV site3H 2-DW as primers and yeast chromosome as template in Table 1 as shown in 159219-159547 of BK006948.2, and has the structure as Genbank: 544490-544789 of BK 006938.2.

Mixing the H1 left homologous arm fragment, the PGK1 promoter fragment, the Hbd gene fragment and the PGK1 terminator fragment in a certain proportion, carrying out PCR reaction by using DNA polymerase produced by Novozam company, annealing at 54 ℃ for 15 seconds, extending for 2min, and after 11 cycles, taking a 1V reaction system as a next PCR reaction template. PCR reaction is carried out by taking Chr IV site3H 1-UP and Chr IV site3 tPGK1-DW in the table 1 as upstream and downstream primers, annealing is carried out for 15 seconds at 54 ℃, the extension time is 2min, 35 cycles are carried out, and a glue recovery kit is utilized to purify a PCR product, so as to obtain the first half part of the Hbd/Crt donor fragment.

After a TP1 promoter, a Crt gene, an Adh1 terminator fragment and a H2 right homologous arm are mixed and dissolved in a certain proportion, DNA polymerase produced by Novozam company is used for PCR reaction, annealing is carried out for 15 seconds at 54 ℃, the extension time is 2min, and after 11 cycles, a 1V reaction system is taken as a next PCR reaction template. PCR reaction is carried out by taking Chr IV site3 pTP1-UP and Chr IV site3H 2-DW in the table 1 as upstream and downstream primers, annealing is carried out for 15 seconds at 54 ℃, the extension time is 2min, 35 cycles are carried out, and a PCR product is purified by using a gel recovery kit. The latter half of the Hbd/Crt donor fragment was obtained.

Mixing the front part and the rear part of the donor fragment in a certain proportion, carrying out PCR reaction by using a Chr IV site3H 1-UP primer pair and a Chr IV site3H 2-DW primer pair in the table 1, annealing at 54 ℃ for 15 seconds, extending for 4min, purifying a PCR product by using a glue recovery kit after 30 cycles to obtain the Hbd/Crt donor fragment

Hbd/Crt donor fragment construction system see Table 2: table 2: Hbd/Crt donor fragment construction system

Thirdly, the method comprises the following steps: BY4741-Hbd-Crt strain construction

The YCplac33-Cas9 plasmid is transformed into a saccharomyces cerevisiae BY4741 competent cell, a positive transformant is screened out BY using a URA3 screening marker, after the positive transformant is cultured to the competent cell, pRS42H-gHbd/Crt plasmid and Hbd/Crt donor fragment are added, the positive transformant is screened out BY using URA3 and a hygromycin screening marker, YPD liquid culture is carried out on the positive transformant, chromosome DNA is extracted and used as a template for PCR verification, and a PCR product is sent for detection reaction. The integration is successful, the insertion position is 544487bp-544489bp of yeast chromosome 4, 3bp of the original genome is replaced BY 3697bp of a Hbd/Crt target gene carrying an expression frame, the insertion position is 544487bp-544489bp of yeast chromosome 4, and the positive transformant strain is named as BY 4741-Hbd-Crt.

Example 2: construction of Yeast Strain inserted with Ter, BCoAT exogenous Gene

Firstly, the method comprises the following steps: construction of pRS42H-gTer/BCoAT plasmid

Ter, the insertion of BCoAT exogenous gene was constructed by using plasmid pRS42H-gTer/BCoAT, the construction process was identical to that of pRS42H-gHbd/Crt plasmid in example 1, the primer pairs used were IPCR-Chr V HIS1-UP and IPCR-Chr V HIS1-DW in Table 1, and the PCR product was 6600bpDNA fragment. The plasmid pRS42H-gTer/BcoAT was obtained. The structure of the gRNA sequence is shown in SEQ ID NO: 78.

II, secondly: Ter/BCoAT Donor fragment construction

The construction of the donor fragment Ter/BCoAT for the insertion of Ter and BCoAT exogenous genes is basically consistent with the construction of the Hbd/Crt donor fragment in the embodiment 1. Nucleotide sequences of Ter gene derived from Treponema dentala and BCoAT gene derived from Faecalibacterium prausnitzii were searched at NCBI and committed against Wikiwisdom organism for codon optimization and gene synthesis. The synthesis system is shown in Table 3. The Ter/BCoAT donor fragment construction system is as follows in table 3:

table 3: Ter/BCoAT donor fragment construction system

In the vector, the structure of the left homology arm of H1 is shown as Genbank: 265076-265364 of CP 020127.1; the Ter gene structure is shown as SEQ ID NO. 3, the BCoAT gene structure is shown as SEQ ID NO. 4, and the H2 right homology arm structure is shown as Genbank: 265373-265594 of CP 020127.1. The promoter and terminator are the same as in example 1.

Thirdly, the method comprises the following steps: BY4741-Hbd-Crt-Ter-BCoAT strain construction

On the basis of the BY4741-Hbd-Crt strain successfully constructed in example 1, the YCplac33-Cas9 plasmid is transformed into Saccharomyces cerevisiae BY4741-Hbd-Crt competent cells, a URA3 screening marker is used for screening out positive transformants, after the positive transformants are cultured to the competent cells, pRS42H-gTer/BCoAT plasmid and Ter/BCoAT donor fragments are added, URA3 and hygromycin screening marker are used for screening out positive transformants, YPD liquid culture is carried out on the positive transformants, chromosome DNA is extracted, PCR verification is carried out BY taking the chromosome DNA as a template, and sequencing verification is carried out on PCR products. The integration is successful, the insertion position is at 265595-265599bp of yeast chromosome 5, 5bp of the original genome is replaced BY 4621bp of Ter/BCoAT target gene carrying an expression cassette, and the positive transformant strain is named as BY 4741-Hbd-Crt-Ter-BCoAT.

Example 3: construction of Yeast strains overexpressing the endogenous Gene of ERG10

Firstly, the method comprises the following steps: construction of pRS42H-gERG10 plasmid

The plasmid pRS42H-gERG10 is used for inserting the endogenous gene of ERG10, the construction process is consistent with the construction of the plasmid pRS42H-gHbd/Crt in example 1, the used primer pairs are IPCR-Chr IV site1-UP and IPCR-Chr IV site1-DW in Table 1, and the PCR product is 6600bpDNA fragment. Plasmid pRS42H-gERG10 was obtained. The structure of the gRNA sequence is shown in SEQ ID NO: 79.

II, secondly: ERG10 Donor fragment construction

Insertion of the endogenous gene of ERG10 the donor fragment ERG10 was constructed. The synthesis system is shown in Table 4.

ERG10 donor fragment constructs are presented in table 4:

table 4: ERG10 donor fragment construction system

In the vector, the structure of the left homology arm of H1 is shown as Genbank: 48865 and 49084 of CP 020126.1; the TEF1 promoter has a structure shown in Genbank: position 699794-700593 of CP 020138.1; the ERG10 gene structure is as Genbank: YPL 028W; the TEF1 terminator has a structure shown in Genbank: position 701971-702470 of CP 020138.1; the structure of the H2 right homology arm is as Genbank: 49093-49392 of CP 020126.1.

Thirdly, the method comprises the following steps: BY4741-Hbd-Crt-Ter-BCoAT-ERG10 strain construction

On the basis of the BY4741-Hbd-Crt-Ter-BCoAT strain successfully constructed in example 2, the YCplac33-Cas9 plasmid is transformed into Saccharomyces cerevisiae BY4741-Hbd-Crt-Ter-BCoAT competent cells, a URA3 screening marker is used for screening out positive transformants, after the positive transformants are cultured to the competent cells, pRS42H-gERG10 plasmid and ERG10 donor fragments are added, URA3 and hygromycin screening markers are used for screening out positive transformants, YPD liquid culture is carried out on the positive transformants, chromosome DNA is extracted and used as a template for PCR verification, and sequencing verification is carried out on PCR products. The integration is successful, the insertion position is at yeast No. 4 chromosome 49085 and 49089bp, 5bp of the original genome is replaced BY 2497bp of an ERG10 target gene carrying an expression cassette, and the positive transformant strain is named as BY4741-Hbd-Crt-Ter-BCoAT-ERG 10.

Example 4: construction of Yeast strains overexpressing the endogenous genes of ERG10, MCT1

Firstly, the method comprises the following steps: construction of pRS42H-gERG10/MCT1 plasmid

The construction of plasmid pRS42H-gERG10/MCT1 for inserting ERG10/MCT1 endogenous genes is consistent with the construction of plasmid pRS42H-gHbd/Crt in example 1, the primer pairs are IPCR-Chr IV site1-UP and IPCR-Chr IV site1-DW in Table 1, and the PCR product is 6600bpDNA fragment. Plasmid pRS42H-gERG10/MCT1 was obtained. The structure of the gRNA sequence is shown in SEQ ID NO: 80.

II, secondly: ERG10/MCT1 Donor fragment construction

The construction of ERG10/MCT1 endogenous gene insertion donor fragment ERG10/MCT1 was essentially identical to the construction of Hbd/Crt donor fragment in example 1. The synthesis system is shown in Table 5.

ERG10/MCT1 donor fragment constructs are described in Table 5:

table 5: ERG10/MCT1 donor fragment construction system

In the vector, the TDH3 promoter has the structure shown in Genbank: position 882012-882811 of CP 020129.1; the MCT1 gene structure is shown as Genbank: the structure of the TDH3 terminator is shown as Genbank 221C: CP020129.1 is shown at positions 883811-884310, which is otherwise the same as in example 3.

Thirdly, the method comprises the following steps: BY4741-Hbd-Crt-Ter-BCoAT-ERG10-MCT1 strain construction

On the basis of the BY4741-Hbd-Crt-Ter-BCoAT strain successfully constructed in example 2, the YCplac33-Cas9 plasmid is transformed into Saccharomyces cerevisiae BY4741-Hbd-Crt-Ter-BCoAT competent cells, a URA3 screening marker is used for screening out positive transformants, after the positive transformants are cultured to the competent cells, pRS42H-gERG10/MCT1 plasmid and ERG10/MCT1 donor fragments are added, the URA3 and hygromycin screening marker are used for screening out positive transformants, YPD liquid culture is carried out on the positive transformants, chromosome DNA is extracted, PCR verification is carried out BY taking the chromosome DNA as a template, and sequencing verification is carried out on PCR products. The integration is successful, the insertion position is at yeast No. 4 chromosome 49085 and 49089bp, 5bp of the original genome is replaced BY 4880bp of ERG10/MCT1 target gene carrying an expression frame, and the positive transformant strain is named as BY4741-Hbd-Crt-Ter-BCoAT-ERG10-MCT 1.

Example 5: construction of MLS1 Gene-deleted Yeast Strain

Firstly, the method comprises the following steps: construction of pRS42H-gMLS1 plasmid

Deletion of MLS1 Gene plasmid pRS42H-gMLS1 was constructed in the same manner as pRS42H-gHbd/Crt plasmid in example 1, the primer pairs used were IPCR-Chr XIV MLS1-UP and IPCR-Chr XIV MLS1-DW in Table 1, and the PCR product was 6600bpDNA fragment. Plasmid pRS42H-gMLS1 was obtained. The structure of the gRNA sequence is shown in SEQ ID NO: 81.

II, secondly: MLS1 deletion Donor fragment construction

Deletion of MLS1 Gene construction of deletion of Donor fragment with MLS1, left homology arm of H1 was synthesized using primer pair Chr XIV MLS1H1-UP (MLS1) and Chr XIV MLS1H 1-DW (MLS1) in Table 1, and right homology arm of H2 was synthesized using primer pair Chr XIV MLS1H 2-UP (MLS1) and Chr XIV MLS1H2-DW (MLS1) in Table 1; after H1 left homologous arm and H2 right homologous arm are mixed and dissolved, PCR reaction is carried out on Chr XIV MLS1H1-UP (MLS1) and Chr XIV MLS1H2-DW (MLS1) by using a primer pair shown in Table 1, and glue recovery and purification are carried out to obtain MLS1 deletion donor fragments, wherein the deletion fragments are deletions of 537bp to 544bp base sequences of MLS1 protein coding sequences and 8bp base sequences.

MLS1 deletion donor fragment constructs refer to table 6:

table 6: MLS1 deletion donor fragment construction System

Primer pair	Form panel	Product of
			P69+P70	BY4741 Strain chromosome	H1 left homology arm, 222bp, in the Donor MLS1 deletion
P71+P72	BY4741 Strain chromosome	H2 right homology arm, 300bp, in the Donor MLS1 deletion
			P69+P72	H1,H2	MLS1 deletion of Donor fragment, 522bp

The sequence information in the vector was the same as in example 4.

Thirdly, the method comprises the following steps: BY4741-Hbd-Crt-Ter-BCoAT-ERG10-MCT 1-delta MLS1 strain construction

The YCplac33-Cas9 plasmid was transformed into the above mentioned Saccharomyces cerevisiae on the basis of successfully constructed strains of examples 2, 3 and 4.

Screening positive transformants from the competent cells by using a URA3 screening marker, culturing the positive transformants to the competent cells, adding pRS42H-gMLS1 plasmid and MLS1 deletion donor fragment, screening the positive transformants by using URA3 and hygromycin screening marker, carrying out YPD liquid culture on the positive transformants, extracting chromosome DNA, carrying out PCR verification by using the chromosome DNA as a template, and carrying out test sending reaction on PCR products. After integration is successful, homologous recombination is utilized, starting from the 536bp position of MLS1 gene fragment, 8bp bases are deleted, and positive transformant strains are named as BY 4741-Hbd-Crt-Ter-BCoAT-delta MLS1, BY4741-Hbd-Crt-Ter-BCoAT-ERG 10-delta MLS1 and BY4741-Hbd-Crt-Ter-BCoAT-MCT 1-delta MLS 1.

Example 6: simultaneously constructs a yeast strain deleting MLS1 gene and overexpressing ACS1 gene

Construction of deletion MLS1 gene and overexpression ACS1 gene yeast strain construction of plasmid pRS42H-gMLS1, the construction process is the same as that of the plasmid construction in example 5. The gRNA sequence has a structure shown in SEQ ID NO: 82.

II, secondly: ACS1 Donor fragment construction

The MLS1 gene is deleted, and the ACS1 gene is over-expressed by constructing the donor fragment ACS1, and the construction process is basically consistent with that of the ERG10 donor fragment in example 3. The synthesis system is shown in Table 7.

ACS1 donor fragment constructs refer to table 7:

table 7: ACS1 donor fragment construction system

In the vector, the structure of the left homology arm of H1 is shown as Genbank: position 315-536 of YNL 117W; the ACS1 gene structure is as Genbank: YAL 054C; the structure of the H2 right homology arm is as Genbank: YNL117W shows 545-844 position, TEF1 promoter and terminator are the same as in example 3.

Thirdly, the method comprises the following steps: construction of BY4741-Hbd-Crt-Ter-BCoAT-ERG10-MCT 1-delta MLS1-ACS1 strain

On the basis of the strains successfully constructed in examples 2, 3 and 4, converting YCplac33-Cas9 plasmid and gRNA into the saccharomyces cerevisiae competent cells, screening out positive transformants by using URA3 screening markers, culturing the positive transformants to the competent cells, adding pRS42H-gMLS1 plasmid and ACS1 donor fragments, screening out the positive transformants by using URA3 and hygromycin screening markers, carrying out YPD liquid culture on the positive transformants, extracting chromosomal DNA, carrying out PCR verification by using the chromosomal DNA as a template, and carrying out sequencing verification on PCR products. The integration is successful, homologous recombination is utilized, original 8bp deletion is carried out at 536bp of MLS1 gene fragment, 3442bp ACS1 gene with an expression frame is successfully integrated, and positive transformation strains are named as BY 4741-Hbd-Crt-Ter-BCoAT-delta MLS1-ACS1, BY4741-Hbd-Crt-Ter-BCoAT-ERG 10-delta MLS1-ACS1, BY4741-Hbd-Crt-Ter-BCoAT-ERG10-MCT 1-delta MLS1-ACS 1.

Example 7: evaluation of butyric acid production ability of various genetically modified yeast strains

The evaluation of butyric acid production by fermentation of the various genetically modified yeast strains is carried out as follows:

1. seed liquid culture, wherein colonies growing on a YPD solid medium plate are selected, inoculated into a test tube filled with 5mL of YPD culture liquid, cultured overnight at 30 ℃ and 220rpm, and subjected to secondary amplification culture if necessary;

2. fermentation, namely a fermentation medium which comprises 10g/L of yeast extract, 20g/L of peptone, 20g/L of glucose and natural pH value; inoculating fresh seed liquid into a 100ml volumetric flask filled with 25ml of fermentation medium, controlling the initial OD6oo value to be about 0.1, and fermenting at 30 ℃ and 220 rpm;

3. pretreatment of fermentation liquor: and (4) acidifying the collected supernatant detection sample. Adding 2mL of supernatant culture solution into a 5mL polyethylene centrifuge tube, adding 0.4mL of 50% sulfuric acid solution and diethyl ether, placing in a shaking table at room temperature and 200rpm for culturing for 45min, then centrifuging at 12000rpm for 5min, taking out the supernatant, placing in another sterile centrifuge tube, adding anhydrous calcium chloride for dehydration, taking out the supernatant, filtering with a filter membrane with the pore diameter of 0.22 mu m, and using the filtrate for gas phase detection;

4. GC-MS gas chromatography conditions: the chromatographic column Agilent 123-; temperature rising procedure: the initial temperature of the column temperature is 60 ℃, the column temperature is kept for 2min, the column temperature is increased to 220 ℃ at the speed of 10 ℃/min, the column temperature is kept for 20min, helium is used as carrier gas, the flow rate is 1mL/min, and the split ratio is 20: 1, the injection volume is 2 μ L, and the initial temperature of the injection port is 250 ℃. Mass spectrum conditions, EI ion source 70eV, ion source temperature 230 ℃, quadrupole temperature 150 ℃, solvent delay time 2min, and scanning mass range m/z 20-150.

5 batches, 144h fermentation production, average butyric acid production is shown in table 8:

table 8: butyric acid production by various genetically modified yeast strains

Name of Strain	Average butyric acid yield (mg/L)
		BY4741	0
BY4741-Hbd-Crt-Ter-BCoAT	193
		BY4741-Hbd-Crt-Ter-BCoAT-ERG10	232
BY4741-Hbd-Crt-Ter-BCoAT-ERG10-MCT1	252
		BY4741-Hbd-Crt-Ter-BCoAT-△MLS1	261
BY4741-Hbd-Crt-Ter-BCoAT-ACS1	270
		BY4741-Hbd-Crt-Ter-BCoAT-△MLS1-ACS1	333
BY4741-Hbd-Crt-Ter-BCoAT-ERG10-MCT1-△MLS1	322
		BY4741-Hbd-Crt-Ter-BCoAT-ERG10-MCT1-△MLS1-ACS1	400

As can be seen from Table 8, the exogenous Hbd/Crt/Ter/BCoAT enables the yeast to have the capability of synthesizing butyric acid, the initial butyric acid yield is 193mg/L on average, but also can directly strengthen the yield of butyric acid by strengthening the metabolic flux of acetoacetyl-CoA and acetyl-CoA, and increases a single gene relative to the modification of the first to the fourth genes, for example, the yield of butyric acid can be obviously increased after the fifth gene modification, the seventh gene modification or the eighth gene modification, the fifth gene modification and the sixth gene modification are increased, the yield of butyric acid is also obviously improved compared with the first gene to the fifth gene, the yield of butyric acid is further improved by the modification of a plurality of genes, in the eight genetically modified strains, the average butyric acid production may be increased to 400mg/L, and in some batches the butyric acid production may even be as high as 465 mg/L.

In the construction strategy of the invention, the butyric acid producing bacteria synthesize butyric acid in the first step, the acetyl coenzyme A in the cell forms butyryl coenzyme A after being catalyzed by a series of enzymes in the central way, the butyryl coenzyme A forms butyric acid in two ways, the traditional way is a butyrate kinase BK way, and the way directly catalyzes the butyryl coenzyme A to remove coenzyme and form butyric acid; the other pathway is a non-BK pathway, namely butyryl-CoA acetate CoA transferase (BCoAT) pathway, which takes butyryl-CoA and acetate as substrates, and the coenzyme is transferred to acetate to form acetyl-CoA under the action of transferase, thereby solving the problem of excessive consumption of acetyl-CoA caused by the synthesis of butyrate, so we select a non-BK pathway to solve the problem of excessive consumption of acetyl-CoA in yeast. The yeast is taken as probiotic bacteria, and the introduced exogenous gene needs to consider that the self probiotic function of the yeast is kept as much as possible under the condition of ensuring the yield of butyric acid, so that bacteria with higher abundance of intestinal butyric acid producing bacteria are selected from the 4 enzyme gene source, the yeast genome is introduced, the probiotic function of the yeast is kept to the greatest extent, and the aim of in vivo treatment of living organisms is fulfilled.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.

Sequence listing

<110> Tianjin university

<120> construction method and application of engineering probiotics

<130> 1

<160> 82

<170> SIPOSequenceListing 1.0

<210> 1

<211> 849

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgaagaagg tttgtgttat tggtgctggt actatgggtt ccggtattgc tcaagctttc 60

gctgctaagg gtttcgaagt tgttttgaga gatattaagg atgaatttgt tgatagaggt 120

ttggatttta ttaacaagaa tttgtccaag ttggttaaga aaggtaaaat tgaagaagct 180

actaaggttg aaattttgac tagaatttct ggtactgttg acttgaatat ggctgctgat 240

tgtgatttgg ttattgaagc tgctgttgaa agaatggata ttaagaagca aattttcgct 300

gatttggata acatttgtaa accagaaact attttggcat ctaatacttc ttctttgtct 360

attaccgaag ttgcttccgc tactaaaact aacgataaag tcattggtat gcacttcttt 420

aacccagctc cagttatgaa gttggttgaa gttattagag gtattgctac ttctcaagaa 480

acatttgatg ccgttaagga aacttctatt gctattggca aggatccagt tgaagttgct 540

gaagctccag gtttcgttgt taacagaatc ttgattccaa tgattaacga agctgttggt 600

attttagctg aaggtattgc ttctgttgaa gatattgata aggctatgaa gttgggtgca 660

aaccatccaa tgggtccttt ggaattgggc gattttatcg gtttggatat ctgtttggct 720

attatggatg ttttatattc tgaaactggc gatagtaaat atagaccaca caccttgttg 780

aagaagtacg ttagagctgg ttggttgggt agaaagtctg gtaaaggttt ctacgattac 840

tctaagtaa 849

<210> 2

<211> 786

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggaattga ataacgttat cttggaaaag gaaggtaaag ttgctgttgt tactattaac 60

agaccaaagg ctttaaacgc tttgaactct gatactttga aggaaatgga ttacgttatt 120

ggtgaaattg aaaatgattc tgaagttttg gctgttattt tgactggtgc tggtgaaaag 180

tccttcgttg ctggtgctga catttctgaa atgaaggaaa tgaatactat tgaaggtaga 240

aagttcggta ttttgggtaa caaggttttc agaagattgg aattgttaga aaagccagtt 300

atcgctgctg ttaacggttt cgctttaggt ggtggttgtg aaattgcaat gtcatgtgat 360

attagaatcg cctcttctaa cgctagattc ggtcaaccag aagttggttt gggtatcact 420

ccaggtttcg gtggtactca aagattgtcc agattggttg gtatgggtat ggctaagcaa 480

ttgatcttta ctgctcaaaa tattaaggct gatgaagctt tgagaattgg tttggttaat 540

aaggttgttg aaccatctga attaatgaac accgctaagg aaattgctaa caagattgtc 600

tctaatgctc cagttgctgt taagttgtct aagcaagcta ttaatagagg tatgcaatgt 660

gatattgata ctgctttagc ttttgaatct gaagcttttg gtgaatgttt ctctactgaa 720

gatcaaaagg atgctatgac tgctttcatt gaaaagagaa agattgaagg ttttaagaat 780

agataa 786

<210> 3

<211> 1194

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgattgtta agccaatggt tagaaacaat atttgtttga atgcccatcc acaaggttgt 60

aagaaaggtg ttgaagatca aattgaatac actaaaaaac gtattactgc tgaagttaaa 120

gccggtgcta aggctccaaa aaacgttttg gtcttgggtt gtagtaatgg ttatggtttg 180

gcttctagaa ttaccgctgc tttcggttac ggtgctgcta ctattggtgt ttctttcgaa 240

aaggccggtt ctgaaaccaa gtacggtacc ccaggttggt acaacaactt ggccttcgac 300

gaagctgcta agagagaagg tttgtactct gttactattg atggcgatgc cttctccgat 360

gaaattaagg ctcaagttat tgaagaagca aagaagaaag gtattaagtt cgatttgatt 420

gtttactcct tggcttctcc agttagaact gatccagata ccggtatcat gcataagtct 480

gttttgaagc cttttggtaa aacattcact ggtaaaactg ttgatccatt cactggtgaa 540

ttgaaggaaa tttctgcaga accagctaat gatgaagaag ctgctgctac tgtcaaggtt 600

atgggtggtg aagattggga acgttggatt aagcaattgt ctaaggaagg tttgttggaa 660

gaaggttgta ttactttagc ttactcttac attggtccag aagctaccca agcattatac 720

agaaagggta ctattggtaa agctaaagaa catttggaag ctaccgccca taggttaaac 780

aaggaaaacc catctattag agctttcgtt tcagtcaaca aaggtttagt tactagagct 840

tctgctgtca ttccagttat tccattgtac ctagcttcct tgttcaaggt tatgaaggaa 900

aaaggtaacc atgaaggttg tattgaacaa attactagat tatacgccga aagattgtac 960

agaaaggacg gtactattcc tgttgatgaa gaaaacagaa tcagaattga tgattgggaa 1020

ttggaagaag atgtccaaaa agctgtctct gctttgatgg aaaaggttac tggtgaaaac 1080

gctgaatctt tgactgattt ggctggttat agacatgatt tcttggcttc caacggtttc 1140

gacgttgaag gtattaacta cgaagctgaa gttgaaagat tcgatagaat ttaa 1194

<210> 4

<211> 1365

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atggatttta ctgaattgta tgctcaaaaa aaaatgactg ctgatcaagc tgctgctttg 60

gttaaatctg gtgattgggt tgattatggt tgggctgtta atactcctgt tgctgttgat 120

gctgctattg ctaaaagatt gcctgaattg gaagatgtta attttagagg tggtattttg 180

atgtgggttc ctgctatttt tcaaattgaa gatcctgctg ctcatatgac ttggaattct 240

tggcatatgg gtgggattga aagaaaagct atagctcaag gtttttcttt ttactctcca 300

attagatatt ctgaattgcc aagatattat agagattctt ctgatcctgt tgatgttgct 360

gtttttcaag ttactccaat ggatgaacat ggttatttta attttggtcc atgtgcttct 420

catttgggtg ccgtttgcga taaggcaaaa aaaattattg tcgaagttaa tagaaatatg 480

ccaaaatgtt tgggtggtac tgaaaattgg gttcatattt ctcaagttgc tggtgttgtt 540

gaaggttcta atccaccaat tggtcaaatg gctgctgctg gtgctgctac tgaagttgat 600

ttgaaagttg ctaatttgat tgttccacaa attcctgatg gtgcttgttt gcaattgggt 660

attggtggta tgccaaatgc tattggtaat ttgattgctc aatctgattt gaaagatttg 720

ggtgttcata ctgaaatgta tgttgatgct tttgttgata ttgctaaagc tggtaaaatt 780

actggtagac ataaaaattt ggataaaggt agacaagttt atgcttttgg tgctggtact 840

caaaaaatgt atgattattt gaatgataat cctgaatgta tggcggctcc tgttgagtac 900

actaacgata ttcgatcgat ctcagccatc gataacttca tttctattaa taacgccgtt 960

gatattgatt tgtttggtca agttaatgct gaatctgctg gtattaaaca tatttctggt 1020

gctggtggtc aattggattt tgttttgggt gcgtacttgt ctaatggcgg aaagtctttt 1080

atttgtcttt catctacttt tatgaataaa aaaactggta aattggaatc tagaattaga 1140

ccaactttgg aaaatggttc tattgttact gatactagag ctaatgttca ttatttgtgt 1200

actgaatatg gttgtgttaa tttgaaaggt ttgacttctt gggaaaaagc tgaagctttg 1260

atttctgttg ctcatcctga ttttagagat gaattgattg ctgaagctga aaaattgcat 1320

atttggagaa gatctaataa aagacatcat catcatcatc attaa 1365

<210> 5

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gcgaagttcg tatattttag ttttctttta ctccaagggt tcgtgacgtc 50

<210> 6

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ggagggaaac acttcctttt tctggccctt acgtatgagc tcatttgccg tctttt 56

<210> 7

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

aaaagacggc aaatgagctc atacgtaagg gccagaaaaa ggaagtgttt ccctcc 56

<210> 8

<211> 55

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cagcaccaat aacacaaacc ttcttcattg ttttatattt gttgtaaaaa gtaga 55

<210> 9

<211> 55

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

tctacttttt acaacaaata taaaacaatg aagaaggttt gtgttattgg tgctg 55

<210> 10

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tgatctatcg atttcaattc aattcaattt acttagagta atcgtagaaa ccttta 56

<210> 11

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

taaaggtttc tacgattact ctaagtaaat tgaattgaat tgaaatcgat agatca 56

<210> 12

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

cagttccatc accattacgc tcccgttagg tacccgggga ctttttttgt tgcaag 56

<210> 13

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

cttgcaacaa aaaaagtccc cgggtaccta acgggagcgt aatggtgatg gaactg 56

<210> 14

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

tttccaagat aacgttattc aattccattt ttagtttatg tatgtgtttt ttgtag 56

<210> 15

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

ctacaaaaaa cacatacata aactaaaaat ggaattgaat aacgttatct tggaaa 56

<210> 16

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

ataaaaatca taaatcatag aaattcgctt atctattctt aaaaccttca atcttt 56

<210> 17

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

aaagattgaa ggttttaaga atagataagc gaatttctat gatttatgat ttttat 56

<210> 18

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

aaatagaaag ctaagaatca aaatcgcaat ccgtgtggaa gaacgattac aacagg 56

<210> 19

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

cctgttgtaa tcgttcttcc acacggattg cgattttgat tcttagcttt ctattt 56

<210> 20

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

aagatataat atttatatat tattattaat attatttttt agttccgaaa 50

<210> 21

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

gttggtgaag gtaaatgtg 19

<210> 22

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

aaacacttcc tttttctggc cctccaaatc ggcaaagtat ttttcagc 48

<210> 23

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

gctgaaaaat actttgccga tttggagggc cagaaaaagg aagtgttt 48

<210> 24

<211> 54

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

gtttctaacc attggcttaa caatcattgt tttatatttg ttgtaaaaag taga 54

<210> 25

<211> 54

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

tctacttttt acaacaaata taaaacaatg attgttaagc caatggttag aaac 54

<210> 26

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

tgatctatcg atttcaattc aattcaattt aaattctatc gaatctttca acttcag 57

<210> 27

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

ctgaagttga aagattcgat agaatttaaa ttgaattgaa ttgaaatcga tagatca 57

<210> 28

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

tcaccattac gctcccgtta ttattgcaga agattagact ttttttgt 48

<210> 29

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

acaaaaaaag tctaatcttc tgcaataata acgggagcgt aatggtga 48

<210> 30

<211> 55

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

ttgagcatac aattcagtaa aatccatttt tagtttatgt atgtgttttt tgtag 55

<210> 31

<211> 55

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

ctacaaaaaa cacatacata aactaaaaat ggattttact gaattgtatg ctcaa 55

<210> 32

<211> 54

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

taaaaatcat aaatcataga aattcgctta atgatgatga tgatgatgtc tttt 54

<210> 33

<211> 54

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

aaaagacatc atcatcatca tcattaagcg aatttctatg atttatgatt ttta 54

<210> 34

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

ccttgtggtc attttttcaa cagatccgtg tggaagaacg attac 45

<210> 35

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

gtaatcgttc ttccacacgg atctgttgaa aaaatgacca caagg 45

<210> 36

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

agcttgtctt caggtgcgt 19

<210> 37

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

gtatgaacca tacgatcagt tttcgtgaac ctggtacgta 40

<210> 38

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

taaagatttc gtgaaccttc cagagtgctt tgataaatcg 40

<210> 39

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

cgatttatca aagcactctg gaaggttcac gaaatcttta 40

<210> 40

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

tcgatacaat gtaaacgttc tgagacattt tgtaattaaa acttagatta gattgc 56

<210> 41

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 41

gcaatctaat ctaagtttta attacaaaat gtctcagaac gtttacattg tatcga 56

<210> 42

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 42

tgcaactaga aaagtcttat caatctcctc atatcttttc aatgacaata gaggaa 56

<210> 43

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 43

ttcctctatt gtcattgaaa agatatgagg agattgataa gacttttcta gttgca 56

<210> 44

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 44

tgttgacgct aacattcaac gctagtatat agcgccgatc aaagtatttg ttacga 56

<210> 45

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 45

tcgtaacaaa tactttgatc ggcgctatat actagcgttg aatgttagcg tcaaca 56

<210> 46

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 46

cttgacctgg gaaggttagt agcttcattt tgtttgttta tgtgtgttta ttcgaa 56

<210> 47

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 47

ttcgaataaa cacacataaa caaacaaaat gaagctacta accttcccag gtcaag 56

<210> 48

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 48

ttaaatgcaa gatttaaagt aaattcactc aatctttgtt ctcctctgcc gccttg 56

<210> 49

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 49

caaggcggca gaggagaaca aagattgagt gaatttactt taaatcttgc atttaa 56

<210> 50

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 50

tccaggtaat attcccaaga ggaatctgtg tatattactg 40

<210> 51

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 51

cagtaatata cacagattcc tcttgggaat attacctgga 40

<210> 52

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 52

cagggcactg tactgcccgt gcctgcgatg agatacatca 40

<210> 53

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 53

gcctccattg agaaatatgc tgatcaacgc tttgaatgct 40

<210> 54

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 54

taaagatttc gtgaaccttc cggggtctca cgataatagt 40

<210> 55

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 55

actattatcg tgagaccccg gaaggttcac gaaatcttta 40

<210> 56

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 56

tgtacggcag agggcgacat tttgtaatta aaacttagat 40

<210> 57

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 57

atctaagttt taattacaaa atgtcgccct ctgccgtaca 40

<210> 58

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 58

gaaaagtctt atcaatctcc ttacaacttg accgaatcaa 40

<210> 59

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 59

ttgattcggt caagttgtaa ggagattgat aagacttttc 40

<210> 60

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 60

aaggtgcttt tccaccatgt atagcgccga tcaaagtatt 40

<210> 61

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 61

aatactttga tcggcgctat acatggtgga aaagcacctt 40

<210> 62

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 62

cactagaatg ttgtcttaat tgatagatga tctcttccat 40

<210> 63

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 63

gcaaatgagc tcatacgtaa gttttagagc tagaaatagc 40

<210> 64

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 64

ttacgtatga gctcatttgc gatcatttat ctttcactgc 40

<210> 65

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 65

aaatactttg ccgatttgga gttttagagc tagaaatagc 40

<210> 66

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 66

tccaaatcgg caaagtattt gatcatttat ctttcactgc 40

<210> 67

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 67

cgatttatca aagcactctg gttttagagc tagaaatagc 40

<210> 68

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 68

cagagtgctt tgataaatcg gatcatttat ctttcactgc 40

<210> 69

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 69

actattatcg tgagaccccg gttttagagc tagaaatagc 40

<210> 70

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 70

cggggtctca cgataatagt gatcatttat ctttcactgc 40

<210> 71

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 71

tccaggtaat attcccaaga atagcgccga tcaaagtatt 40

<210> 72

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 72

aatactttga tcggcgctat tcttgggaat attacctgga 40

<210> 73

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 73

gcctccattg agaaatatgc tgat 24

<210> 74

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 74

taaagatttc gtgaaccttc cggggtctca cgataatagt 40

<210> 75

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 75

atctaagttt taattacaaa acatggtgga aaagcacctt 40

<210> 76

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 76

cactagaatg ttgtcttaat tg 22

<210> 77

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 77

gcaaatgagc tcatacgtaa 20

<210> 78

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 78

aaatactttg ccgatttgga 20

<210> 79

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 79

cgatttatca aagcactctg 20

<210> 80

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 80

cgatttatca aagcactctg 20

<210> 81

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 81

actattatcg tgagaccccg 20

<210> 82

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 82

actattatcg tgagaccccg 20

Claims

1. An engineered probiotic comprising a modification of four exogenous genes for butyrate metabolizing enzymes, said four exogenous genes comprising: the first gene encodes 3-hydroxybutyryl-CoA dehydrogenase protein (HBD), the second gene encodes enoyl-CoA hydratase protein (CRT), the third gene encodes trans-2-enoyl-CoA reductase protein (TER), and the fourth gene encodes butyryl-CoA: acetate CoA transferase protein (BCoAT).

2. The engineered probiotic bacteria of claim 1, further comprising a fifth, sixth, seventh and/or eighth genetic modification, wherein the fifth and/or sixth genetic modification increases the positive regulation of the synthesis of the precursor acetoacetyl-CoA in the butyrate synthesis pathway, and the seventh and/or eighth genetic modification increases acetoacetyl-CoA by increasing the regulation of acetyl-CoA,

preferably, the fifth and/or sixth gene comprises the engineered probiotic endogenous proteins acetyl-coenzyme A C-acetyltransferase ERG10 and/or malonyl-coenzyme a: ACP transferase protein MCT1 encoding gene, the seventh gene includes malate synthase protein MLS1 encoding gene, the eighth gene includes acetyl-CoA synthetase protein ACS1 encoding gene.

3. The engineered probiotic bacteria of any one of claims 1 to 2, wherein the first to sixth and eighth genetic modifications increase or overexpress the activity of the encoded enzyme, and the seventh genetic modification decreases or represses the activity or expression of the encoded enzyme, preferably wherein the genetic modifications increase or overexpress the activity of the encoded enzyme comprise point mutations, strong promoters, enhancers, copy number increase or fusion co-expression, and the genetic modifications decrease or represses the activity or expression of the encoded enzyme comprise point mutations, deletions, insertions, antisense polyneotides, siRNA, microRNA, CRISPR.

4. The engineered probiotic of any of claims 1 to 3, wherein said probiotic comprises yeasts, probiotic spores, Clostridium butyricum, Lactobacillus, Bifidobacterium, Actinomycetes, preferably said yeasts comprise any of Saccharomyces cerevisiae (Saccharomyces cerevisiae), Saccharomyces pastorianus (Pichia pastoris), Pichia stipitis (Pichia pastoris), Saccharomyces Bayanus and Candida shehatae (Candida shehatae).

5. A method of constructing engineered probiotic bacteria according to any one of claims 1 to 4, wherein said method of construction comprises: introducing modification of four exogenous genes of butyrate metabolic enzyme into engineering probiotics, so that the engineering probiotics sequentially react and synthesize 3-hydroxybutyryl coenzyme A, crotonyl coenzyme A, butyryl coenzyme A and butyrate on the basis of taking acetoacetyl coenzyme A as a substrate, and complete 4-step reaction of synthesizing butyrate, wherein the four exogenous genes comprise: the first gene encodes 3-hydroxybutyryl-CoA dehydrogenase protein (HBD), the second gene encodes enoyl-CoA hydratase protein (CRT), the third gene encodes trans-2-enoyl-CoA reductase protein (TER), and the fourth gene encodes butyryl-CoA: acetate CoA transferase protein (BCoAT).

6. The method of construction according to claim 5, comprising introducing a fifth gene, a sixth gene, a seventh gene, and/or an eighth gene modification in the engineered probiotic.

7. A method for producing butyric acid using the engineered probiotic of any of claims 1 to 4, comprising culturing said engineered probiotic in a culture medium.

8. A fermentation product produced by fermentation using the engineered probiotic of any one of claims 1 to 4.

9. Use of the engineered probiotic of any one of claims 1 to 4 or the fermentation product of claim 8 for the production of butyric acid.

10. Use of the engineered probiotic bacteria of any one of claims 1 to 4 or the fermentation product of claim 8 in the fields of medicine, animal husbandry, food, health care or chemical engineering, preferably said use comprises use as feed or feed additive for animal husbandry.

11. A formulation comprising the engineered probiotic of any one of claims 1 to 4 or the fermentation product of claim 8.

12. The formulation according to claim 11, wherein the engineered probiotic bacteria or the fermentation product is further processed into any other dosage form, preferably the dosage form is an edible dosage form, more preferably the dosage form is in the form of a powder, granules, tablets, capsules, or a liquid.