Engineered fungi and methods for their construction and use

Abstract

We provide an engineered fungus capable of increasing tryptophan yield, as well as methods for its construction and use. Through screening, we isolated a strain capable of tolerating high concentrations of tryptophan. After analyzing its genome and protein sequences, we discovered that point mutations occurred in several proteins in the strain, and these mutants were able to increase tryptophan yield. To further increase tryptophan yield, we modified the protein sequence expressed by the fadR gene or the protein sequence expressed by the pepD gene in the starting strain. Through these modifications, the resulting engineered fungus exhibited a higher tryptophan yield than the starting strain. In large-scale production, the tryptophan yield in a 5L fermenter reached 62.38±5.80 g / L, with a sugar acid conversion rate of 24.1%. Compared to the original strain, the tryptophan yield increased 1.48-fold and the sugar acid conversion rate increased 1.26-fold. The above biomaterials and their uses belong to the field of molecular biology and have broad practical value.

Description

[Technical Field]

[0001] The present invention relates to the technical field of molecular biology, and in particular to engineered bacteria and methods for their construction and use.

[0002] [Incorporated by reference] This application claims priority from Chinese Application No. 202311807065.2 filed on December 26, 2023, the entire contents of which are incorporated herein by reference. [Background technology]

[0003] L-tryptophan is an essential amino acid widely used in food, feed, and pharmaceutical applications. Tryptophan biosynthesis mainly involves three modules: the central carbon metabolic pathway (CCM), the shikimic acid (SHIK) pathway, and the branched acid (CHA) pathway. However, its production pathway is long, requires many precursors, and has strong feedback inhibition, resulting in relatively low production efficiency. Consequently, the yield of industrially mature strains is difficult to meet market demand. With the ongoing development of synthetic biology, an increasing number of metabolic engineering strategies are being used to construct cell factories that efficiently produce L-tryptophan. However, while rational engineering of metabolic pathways has clear objectives and significant benefits, due to the complex nature of microorganisms, previous reports have shown that the expression of cytidine deaminase only slightly increases the mutation rate. Therefore, existing approaches to metabolic network engineering cannot rapidly increase yield. Summary of the Invention [Means for solving the problem]

[0004] To solve the above problems, the present invention screens bacterial strains for resistance to high concentrations of tryptophan, identifies strains that can tolerate high concentrations of tryptophan, and then analyzes the genome and protein sequences of the strains. Point mutations were found in several proteins in the strain, and these mutants were found to increase tryptophan yield. The present invention induces mutations in the strains that are not related to the main metabolic pathway for amino acid synthesis and the glycolytic pathway, resulting in strains with superior properties. However, due to the high complexity of biological metabolic networks, the mutation results obtained by the present invention are highly uncertain, making accurate prediction and control difficult at the early stage of research. Those skilled in the art typically do not consider whether modifying these proteins is beneficial for tryptophan production. Based on the suggestion of a mutation system fusing cytidine deaminase with T7 RNA polymerase, the applicant discovered that fusing cytidine deaminase with the α subunit of E. coli RNA polymerase can accelerate mutations to a level that supports effective adaptive evolution of E. coli without reducing cellular activity. Guided evolution is a means for the industry to rapidly improve the production capacity of bacterial strains. Combined with advanced technologies such as genome sequence analysis, it combines production capacity improvement with principle exploration, providing the basis and ideas for subsequent rational modification, and can improve the production level of bacterial strains and strengthen their international competitiveness.

[0005] In a first aspect, the present invention claims an engineered fungus with increased tryptophan yield, wherein a starting strain is engineered to obtain the engineered fungus with increased tryptophan yield. In some embodiments, the engineered fungus comprises a gene encoding at least one of a pepD mutant protein and a fadR mutant protein. In a second aspect, the present invention claims a method for constructing an engineered fungus with increased tryptophan yield. In some embodiments, the method comprises the step of engineering at least one of a pepD protein and a fadR protein of a starting strain to obtain the engineered fungus, wherein, under the same culture conditions, the engineered fungus has a higher tryptophan yield than the starting strain.

[0006] In a third aspect, the present invention claims a biomaterial for increasing the yield of tryptophan.

[0007] In some embodiments, the biological material comprises a mutant protein, wherein the mutant protein comprises a sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:1 or a sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:2.

[0008] In some embodiments, the biological material comprises a DNA molecule, the DNA molecule comprising a gene encoding the mutant protein.

[0009] In some embodiments, the biological material comprises a gene expression cassette, the gene expression cassette comprising the mutant protein or a gene encoding the mutant protein.

[0010] In some embodiments, the biological material comprises a recombinant vector, the recombinant vector comprising the mutant protein or a gene encoding the mutant protein.

[0011] In a fourth aspect, the present invention claims the use of engineered fungi in increasing the yield of tryptophan.

[0012] In the present invention, the production of tryptophan specifically means that the strain is capable of producing and accumulating tryptophan when cultured in a culture medium.

[0013] As used herein, the term "protein of the invention" has the meaning commonly understood by those of skill in the art.

[0014] The "pepD protein mutant" of the present invention can be obtained by mutating the amino acid sequence shown in SEQ ID NO:4. Specifically, the pepD protein mutant of the present invention has the amino acids at positions 21, 225, and 484 of the amino acid sequence shown in SEQ ID NO:4 substituted with threonine, alanine, and lysine, respectively. Also included within the scope of the present invention are enzymes derived from E. coli that have more than 80%, preferably 90%, more preferably 95%, and most preferably 99% or more identity with SEQ ID NO:1 and have the function of degrading dipeptides with unblocked N-terminus.

[0015] Similarly, the "fadR protein mutant" of the present invention can be obtained by mutating the amino acid sequence shown in SEQ ID NO:5. Specifically, the fadR protein mutant of the present invention has threonine and isoleucine substituted at positions 140 and 171, respectively, corresponding to the amino acid sequence shown in SEQ ID NO:5. Also included within the scope of the present invention are proteins derived from Escherichia coli that have greater than 80%, preferably 90%, more preferably 95%, and most preferably 99% or more identity with SEQ ID NO:2 and have dual DNA-binding transcriptional regulator activity, which are also included within the scope of the present invention.

[0016] The term "exogenous" as used herein refers to the inclusion of a substance that is not originally present in a system. For example, when an enzyme that is not originally present in a strain is expressed in the strain by introducing a gene encoding the enzyme into the strain by a method such as transformation, the enzyme is considered to be "exogenous" to the strain, but this is not limited thereto.

[0017] The term "enhancement" as used herein refers not only to an effect of increasing the activity of the protein itself to be higher than its original function, but also to at least one method selected from the group consisting of increasing the copy number of nucleotides encoding the protein, modifying the regulatory sequence of the gene encoding the protein, replacing the regulatory sequence of the gene encoding the protein on a chromosome with a sequence having strong activity, replacing the gene encoding the protein with a mutant gene to increase the activity of the protein, and introducing a modification into the gene encoding the protein on a chromosome to increase the activity of the protein. The term "enhancement" includes, but is not limited to, any existing method as long as it can increase the activity of the protein or the activity of the introduced protein compared to the endogenous activity.

[0018] The term "introduced protein activity" as used herein has the meaning commonly understood by those skilled in the art and can be achieved by methods known in the art, including, but not limited to, for example, inserting a polynucleotide comprising a polynucleotide sequence encoding a protein into a chromosome, and / or cloning the polynucleotide into a vector to introduce the polynucleotide into a microorganism, and / or directly increasing the copy number of the polynucleotide on the chromosome, and / or modifying a promoter carrying a polynucleotide encoding the protein to increase the transcription initiation rate, and / or modifying the transcription of the polynucleotide encoding the protein to increase its activity, and / or modifying the translation regulatory sequence of a messenger RNA carrying a polynucleotide encoding the protein to increase translation strength, and / or modifying the polynucleotide encoding the protein itself to increase mRNA stability, protein stability, and release feedback inhibition of the protein, and the like, including, but not limited to, any known method by which protein activity can be introduced.

[0019] A vector is a DNA construct containing a polynucleotide sequence encoding a target protein, operably linked to an appropriate regulatory sequence so that the target protein can be expressed in a host cell. After being introduced into a suitable host cell, the vector may replicate or function independently of the host cell genome, or may be integrated into the host genome. These vectors are not particularly limited as long as they are replicable in the host cell. Examples of vectors include natural or recombinant plasmids, cosmids, viruses, and phages. For example, pWE15, pET, and pUC vectors are included. Alternatively, by inserting a vector into the host cell chromosome, the polynucleotide encoding the endogenous target protein on the chromosome can be replaced with a modified polynucleotide. Insertion of a polynucleotide into a chromosome can be carried out using any method known in the art, including, but not limited to, homologous recombination. The polynucleotide can include DNA and RNA encoding the target protein and can be inserted into the host cell chromosome in any form as long as it can be expressed in the host cell. For example, the polynucleotide can be introduced into the host cell in its original state and / or in the form of an expression cassette. An expression cassette is a genetic construct that contains all the essential elements required for autonomous expression, and may be an autonomously replicating expression vector, and may include a promoter, a transcription termination signal, a ribosome binding domain, and a translation termination signal operably linked to a polynucleotide.

[0020] Similarly, the term "reduction" as used herein refers to reducing, weakening, diminishing, or completely eliminating the activity of a protein, such as an enzyme. In specific embodiments, reducing the activity of an enzyme can be achieved by methods such as, but not limited to, knocking out part or all of the gene encoding the enzyme, inactivating or partially inactivating mutations in the gene, reducing transcription or translation by modifying the gene promoter or translational regulatory region, modifying the gene sequence to reduce mRNA stability or destabilize the enzyme structure, or regulating the gene with sRNA, or a combination thereof.

[0021] The term "host cell" as used herein has the meaning commonly understood by those skilled in the art, i.e., a bacterial strain containing a protein or a mutant thereof. In other words, the present invention may use any host cell containing a target protein or a mutant thereof and capable of producing tryptophan. The host cell may preferably be derived from Escherichia coli (E. coli). Specifically, the host referred to in the present invention refers to a strain capable of producing tryptophan, i.e., a bacterium capable of producing tryptophan when cultured in a broth, capable of accumulating tryptophan, or capable of secreting tryptophan into the medium, i.e., obtaining extracellular free tryptophan, and particularly capable of accumulating more tryptophan than a wild-type strain or parent strain. To confer the ability to produce tryptophan to a strain, conventional breeding methods, such as culturing auxotrophic mutant strains, analog-resistant strains, or metabolically controlled mutant strains capable of producing tryptophan, and recombinant strains with increased activity of enzymes involved in amino acid biosynthesis, or a combination of the above methods, can be used.

[0022] The term "containing the pepD and / or fadR protein mutant of the present invention" as used herein has the meaning commonly understood by those skilled in the art and can be achieved by methods known in the art, including, but not limited to, for example, inserting a polynucleotide containing a polynucleotide sequence encoding the protein into a chromosome, and / or cloning the polynucleotide into a vector to introduce into a microorganism, and / or directly increasing copies of the polynucleotide on the chromosome, and includes, but is not limited to, any known method capable of introducing protein activity.

[0023] Those skilled in the art know that it is more important to mutate wild-type polypeptides to increase activity and find sites that can achieve desired goals. Therefore, based on the teachings of the present invention, those skilled in the art can replace the serine at position 21 with threonine, the glycine at position 225 with alanine, the alanine at position 484 with lysine in the amino acid sequence represented by the pepD protein, and the alanine at position 140 with threonine and the leucine at position 171 with isoleucine in the amino acid sequence represented by the fadR protein, and detect the relative activity of the mutants.

[0024] In addition, those skilled in the art will readily recognize that changing a small number of amino acid residues in some regions of a polypeptide, e.g., in non-critical regions, will not significantly alter biological activity, and for example, a sequence obtained by appropriately replacing a few amino acids will not affect its activity (see Watson et al., Molecular Biology of the Gene, 4th ed., 1987, The Benjamin / Cummings Pub. Co., p. 224). Therefore, those skilled in the art can make such substitutions and ensure that the resulting molecule still has the desired biological activity.

[0025] Therefore, it is clear that the pepD and / or fadR proteins and their mutants of the present invention can be further mutated to obtain additional mutants that still have the corresponding functions and activities. For example, those skilled in the art know that adding or deleting several amino acid residues, e.g., preferably 1 to 20, more preferably 1 to 15, more preferably 1 to 10, more preferably 1 to 3, and most preferably 1, at any end of a polypeptide does not affect the function of the resulting mutant. For example, to facilitate purification, engineers often attach a 6xHis tag to either end of the resulting protein, but such a protein has the same function as a protein without the 6xHis tag. Therefore, the present invention should include conservative mutants obtained based on the present invention. [Effects of the Invention]

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention involves modifying the protein sequence expressed by the fadR gene or the pepD gene in a starting strain to obtain an engineered bacterium with increased tryptophan yield compared to the starting strain. The starting strain is a tryptophan-producing strain, and the modification of the fadR gene reduces or even knocks out its expression level or protein activity, while the modification of the pepD gene increases its expression level or protein activity. In large-scale production, the tryptophan yield in a 5L fermenter reached 62.38±5.80 g / L, with a sugar acid conversion rate of 24.1%. Compared to the original strain, the tryptophan yield increased by 1.48-fold, and the sugar acid conversion rate increased by 1.26-fold.

[0028] The deposit information for the engineered fungi of the present invention is as follows:

[0029] Escherichia coli IBEWQ-624 was deposited at the China Typical Culture Depository Center on August 19, 2024, with the deposit number CCTCC NO: M20241821 and the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. [Brief explanation of the drawings]

[0030] [Figure 1] Figure 1 shows the tryptophan fermentation levels of strain IBEWQ-62 and the strains after different degrees of fadR reduction and / or different degrees of pepD enhancement. The results show that enhanced expression of pepD gene significantly improved fermentation efficiency. The optimal solution for reducing fadR expression is knockout of the fadR gene. DETAILED DESCRIPTION OF THE INVENTION

[0031] The engineered bacteria according to one embodiment of the present invention are modified from a starting strain and contain genes encoding at least one of a pepD mutant protein and a fadR mutant protein.

[0032] In some embodiments, the pepD mutant protein comprises at least one of the following mutations: S21T, G225A, and A484K. For example, the mutation in the pepD mutant protein may be S21T, G225A, or A484K, each of which is a pepD mutation. S21T , pepD G225A or pepD A484K For example, the mutations in the pepD mutant protein may be S21T and G225A, G225A and A484K, or S21T and A484K, respectively. S21T,G225A , pepD G225A,A484K or pepD A484K,S21T It is expressed as:

[0033] In some embodiments, the mutations in the pepD mutant protein are S21T, G225A, and A484K mutations, and A484K,S21T,G225A It is expressed as:

[0034] where pepD S21T shows that the serine at position 21 of the amino acid sequence of the pepD protein (SEQ ID NO:4) is replaced with a threonine to detect the related activity of the protein mutant.

[0035] pepD G225A shows that the glycine at position 225 of the amino acid sequence of the pepD protein is replaced by an alanine to detect the related activity of the protein mutant.

[0036] pepD A484K shows that the alanine at position 484 of the amino acid sequence of the pepD protein is replaced by a lysine to detect the associated activity of the protein mutant.

[0037] In some embodiments, the pepD mutant protein comprises a sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the pepD mutant protein comprises a sequence having at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the pepD mutant protein comprises a sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the pepD mutant protein comprises a sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the pepD mutant protein comprises a sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:1.

[0038] In some embodiments, the pepD mutant protein has the amino acid sequence shown in SEQ ID NO:1.

[0039] In some embodiments, the fadR mutant protein comprises at least one of the following mutations: A140T and L171I. For example, the mutation in the fadR mutant protein may be an A140T or L171I mutation, and the fadR A140T or fadR L171I and may have A140T and L171I mutations, and fadR A140T,L171I It is expressed as:

[0040] where fadR A140T indicates that the alanine at position 140 of the amino acid sequence shown in the fadR protein (SEQ ID NO:5) is replaced with threonine to detect the relative activity of the protein mutant.

[0041] fadR L171Ishows that the leucine at position 171 of the amino acid sequence shown in the fadR protein is replaced with isoleucine to detect the related activity of the protein mutant.

[0042] In some embodiments, the fadR mutant protein comprises a sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the fadR mutant protein comprises a sequence having at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the fadR mutant protein comprises a sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the fadR mutant protein comprises a sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the fadR mutant protein comprises a sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:2.

[0043] In some embodiments, the engineered bacterium comprises a pepD mutant protein and a fadR mutant protein, wherein the pepD mutant protein comprises S21T, G225A, and A484K mutations, and the fadR mutant protein comprises A140T and L171I mutations.

[0044] In some embodiments, the pepD mutant protein has the amino acid sequence set forth in SEQ ID NO:1 and the fadR mutant protein has the amino acid sequence set forth in SEQ ID NO:2.

[0045] As used herein, an engineered strain is one that has been modified from a starting strain. The starting strain is the original strain used for breeding. For example, the starting strain may be derived from a spontaneously mutated strain during production, or from a strain that has advantageous properties for further research or use, such as fast growth and low nutritional requirements. For example, the starting strain may be a strain that has already undergone other mutations, or a mutant strain that is highly sensitive to inducers.

[0046] In some embodiments, the starting strain is any one selected from Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, and yeast cells.

[0047] In some embodiments, the starting strain is one selected from strain IBEWQ, mutant strain IBEWQ-62, mutant strain IBEWQ-624, E. coli Nissle1917, E. coli BL21, E. coli HB101, E. coli JM109, E. coli DH10B, or E. coli MG1655.

[0048] In some embodiments, the modification comprises increasing the expression level of the pepD protein of the starting strain.

[0049] In some embodiments, the modification comprises reducing the expression level of FadR protein in the starting strain.

[0050] In some embodiments, the modification involves knocking out the fadR gene in the genome of an engineered strain that contains a pepD mutant protein containing S21T, G225A, and A484K mutations and a fadR mutant protein containing A140T and L171I mutations, and replacing the promoter of the gene encoding the pepD mutant protein in the genome of the starting strain with a strong promoter.

[0051] In the examples herein, the engineered strains exhibit increased tryptophan yields compared to the unengineered starting strain under the same culture conditions.

[0052] Examples herein provide methods for constructing an engineered bacterium with a high tryptophan yield, and in some examples, the method comprises modifying at least one of the pepD protein and the fadR protein of a starting strain to obtain the engineered bacterium, wherein under the same culture conditions, the modified engineered bacterium has a higher tryptophan yield than the starting strain.

[0053] In some embodiments, the modification includes mutating an amino acid site of the pepD protein of the starting strain, including at least one of the mutations S21T, G225A, and A484K, to generate a pepD mutant protein; overexpressing a gene encoding the pepD protein or the pepD mutant protein using a high-copy plasmid as a vector; replacing the promoter of the gene encoding the pepD protein or the pepD mutant protein in the genome of the starting strain with a strong promoter; or improving the stability of mRNA transcribed from the gene for the pepD protein or the pepD mutant protein.

[0054] In some embodiments, the pepD mutant protein comprises a sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:1.

[0055] In some embodiments, the modification includes knocking out the gene encoding the fadR protein of the starting strain, or mutating the amino acid site of the fadR protein of the starting strain, including at least one of the A140T and L171I mutations, to generate a fadR mutant protein, or replacing the promoter of the gene encoding the FadR protein or the FadR mutant protein in the genome of the starting strain with a weak promoter, or suppressing the translation efficiency or reducing the stability of mRNA transcribed from the gene for the FadR protein or the FadR mutant protein. In some embodiments, the fadR mutant protein comprises a sequence having at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO:2.

[0056] In some embodiments, the modification includes directly modifying the endogenous pepD and / or fadR proteins of the starting strain, or modifying and introducing exogenous pepD and / or fadR proteins into the starting strain, wherein the modification method is selected from at least one of the following technical means:

[0057] a) In the starting strain, the modifications made to the pepD protein may be any of the following:

[0058] (a1) Three amino acid mutations were performed on the pepD protein, namely S21T, G225A, and A484K, to obtain a mutant protein pepD, hereinafter referred to as the mutant protein pepD. S21T,G225A,A484K and its amino acid sequence is shown in SEQ ID NO:1.

[0059] (a2) Using a high-copy plasmid as a vector, the pepD protein or pepD S21T,G225A,A484K The gene encoding the mutant protein is overexpressed.

[0060] (a3) the pepD protein or pepD of the genome S21T,G225A,A484K The promoter of the gene encoding the mutant protein is replaced with a strong promoter.

[0061] (a4) pepD protein or pepD S21T,G225A,A484K Improves the stability of mRNA transcribed from the gene encoding the mutant protein.

[0062] (a5) pepD protein or pepD S21T,G225A,A484K Any method and combination thereof that can upregulate the expression level of the gene encoding the mutant protein.

[0063] b. In the starting strain, the fadR protein may be modified in any of the following ways:

[0064] (b1) Knock out the gene encoding the fadR protein in the starting strain.

[0065] (b2) Two nucleotide mutations were made in the fadR protein-encoding gene, A140T and L171I, respectively, to obtain a mutant fadR protein, hereinafter referred to as mutant protein FadR. A140T,L171I and its amino acid sequence is shown in SEQ ID NO:2.

[0066] (b3) FadR protein or FadR A140T,L171I The promoter of the gene encoding the mutant protein is replaced with a weak promoter.

[0067] (b4) FadR protein or FadR A140T,L171I The translation efficiency of mRNA transcribed from the gene encoding the mutant protein is suppressed or its stability is reduced.

[0068] (b5) FadR protein or FadR A140T,L171IAny method and combination thereof that can reduce the expression level of the gene encoding the mutant protein.

[0069] Furthermore, in a specific embodiment of the present invention, the starting strain is a tryptophan-producing strain, and the starting strain is any one selected from Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, yeast cells, etc., and when the starting strain is Escherichia coli, it is more preferably one selected from strain IBEWQ, mutant strain IBEWQ-62, mutant strain IBEWQ-624, E. coli Nissle1917, E. coli BL21, E. coli HB101, E. coli JM109, E. coli DH10B, or E. coli MG1655.

[0070] In some embodiments, the engineered fungus is mutant strain IBEWQ-624. Mutant strain IBEWQ-624 comprises a pepD mutant protein and a fadR mutant protein, wherein the pepD mutant protein has the amino acid sequence set forth in SEQ ID NO:1 and the fadR mutant protein has the amino acid sequence set forth in SEQ ID NO:2.

[0071] In some embodiments, the step of obtaining the engineered fungus includes the steps of culturing the starting strain in a culture medium containing different concentrations of tryptophan and detecting the biomass in the culture medium, determining the growth rate of the starting strain based on the biomass, and fermenting the strain that has a faster growth rate in the culture medium with a high tryptophan concentration to obtain the engineered fungus capable of increasing tryptophan yield.

[0072] In some embodiments, the concentration of the high-concentration tryptophan is 50 g / L to 70 g / L.

[0073] In a specific embodiment of the present invention, the engineered strain IBEWQ-624 has a genome in which the gene encoding the fadR protein is knocked out and the gene encoding the pepD protein is inserted into the genome of the IBEWQ strain. S21T,G225A,A484KMutations were made in the protein-encoding gene, and pepD S21T,G225A,A484K The promoter of the gene is replaced with the strong promoter PJ23119.

[0074] The mutant protein according to one embodiment of the present specification comprises a sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:1 or a sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:2.

[0075] In some embodiments, the mutant protein comprises a pepD mutant protein, wherein the pepD mutant protein comprises at least one of the following mutations: S21T, G225A, and A484K.

[0076] In some embodiments, the mutant protein comprises a fadR mutant protein, wherein the fadR mutant protein comprises at least one of the following mutations: A140T and L171I.

[0077] One of the examples herein provides a DNA molecule, and in some examples, the DNA molecule comprises a gene encoding the mutant protein.

[0078] One embodiment of the present specification provides a gene expression cassette, and in some embodiments, the gene expression cassette comprises the mutant protein or a gene encoding the mutant protein.

[0079] One embodiment of the present specification provides a recombinant vector, and in some embodiments, the recombinant vector comprises the mutant protein or a gene encoding the mutant protein.

[0080] One of the examples herein provides the use of engineered fungi in increasing the yield of tryptophan.

[0081] In some embodiments, the use comprises fermenting the engineered fungus to obtain an increased yield of tryptophan.

[0082] In some embodiments, the pepD mutant protein contained in the engineered bacterium is a protein (A1) whose amino acid sequence is SEQ ID NO:1, or A protein (A2) in which one or more amino acid residues are substituted and / or deleted and / or added to the amino acid sequence shown in SEQ ID NO: 1 and has the same function as the amino acid sequence shown in SEQ ID NO: 1; or A protein (A3) having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity with the amino acid sequence defined by any one of (A1) to (A2) and having the same function; or It includes any one of fusion proteins (A4) obtained by linking a tag to the N-terminus and / or C-terminus of a protein defined by any one of (A1) to (A3).

[0083] In some embodiments, the fadR mutant protein contained in the engineered bacterium includes any one of a protein (A1) whose amino acid sequence is SEQ ID NO:2; a protein (A2) having the same function as the amino acid sequence shown in SEQ ID NO:2, with one or more amino acid residues substituted and / or deleted and / or added; a protein (A3) having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity with the amino acid sequence defined by any one of (A1) to (A2), with the same function; and a fusion protein (A4) obtained by linking a tag to the N-terminus and / or C-terminus of a protein defined by any one of (A1) to (A3).

[0084] In some embodiments, the gene encoding the pepD mutant protein is pepD S21T,G225A,A484K These include DNA molecules that have 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to the DNA sequence defined by the gene encoding the mutant protein.

[0085] In some embodiments, the gene encoding the fadR mutant protein is fadR A140T,L171I These include DNA molecules that have 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to the DNA sequence defined by the gene encoding the mutant protein.

[0086] The present invention claims any of the following biomaterials for increasing the yield of tryptophan:

[0087] (I) Protein: pepD S21T,G225A,A484K Mutant protein and / or fadR A140T,L171I Mutant proteins.

[0088] (II) Gene: pepD S21T,G225A,A484K Mutant protein and / or fadR A140T,L171I The gene encoding the mutant protein.

[0089] (III) Expression cassette: pepD as described above S21T,G225A,A484K Mutant protein and / or fadR A140T,L171I An expression cassette containing a gene encoding a mutant protein or an expression cassette containing the above DNA fragment.

[0090] (IV) Recombinant vector: pepD S21T,G225A,A484K Mutant protein and / or fadR A140T,L171I A recombinant vector containing a gene encoding a mutant protein or a recombinant vector containing the above DNA fragment.

[0091] (V) Recombinant bacteria: pepD S21T,G225A,A484K Mutant protein and / or fadR A140T,L171I A recombinant bacterium containing a gene encoding a mutant protein or a recombinant bacterium containing the above DNA fragment.

[0092] (a) Use of biomaterials in increasing the tryptophan yield of starting strains.

[0093] (b) Use of biomaterials in the production of tryptophan.

[0094] (c)pepD S21T,G225A,A484K Mutant protein and / or fadR A140T,L171I Use of mutant proteins in increasing the tryptophan yield of a starting strain.

[0095] (d) pepD S21T,G225A,A484K Mutant protein and / or fadR A140T,L171I Use of mutant proteins in the production of tryptophan.

[0096] Example Hereinafter, specific embodiments of the present invention will be described in detail, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments.

[0097] In the present invention, unless otherwise specified, all experimental methods used are conventional methods, and all materials, reagents, etc. used can be purchased commercially.

[0098] Example 1 Screening of resistant strains that grow faster in tryptophan fermentation broth

[0099] Fusion of cytidine deaminase with the α subunit of Escherichia coli RNA polymerase can accelerate mutations to levels that support efficient adaptive evolution in Escherichia coli without compromising cellular activity.

[0100] The gene sequences for the RNA polymerase α subunit and cytidine deaminase genes on the genome of the starting strain were amplified using primers RNAP α-F / R and CDA-F / R. The sequences at both ends were then fused and expressed using RNAP α-F and CDA-R as primers. The resulting gene was then double-cleaved with EcoRI and NcoI enzymes, and ligated into the similarly double-cleaved temperature-sensitive plasmid pKD46 (GenBank accession no.: MF287367). The resulting plasmid, designated pKAP, contained an arabinose-inducible promoter to control the expression of the RNA polymerase α subunit and cytidine deaminase genes.

[0101] Primer sequences used in the construction of plasmid pKAP.

[0102] RNAP α-F:GAATTCatgcagggttctgtgacag, SEQ ID NO:7.

[0103] RNAP α-R:CGATCCGCCACCGCCAGAGCCACCTCCGCCctcgtcagcgatgcttgccggtg, SEQ ID NO:8.

[0104] CDA-F:GGCGGAGGTGGCTCTGGCGGTGGCGGATCGcatccacgttttcaaaccgc, SEQ ID NO:9.

[0105] CDA-R:CCATGGttaagcgagaagcactcgg, SEQ ID NO:10.

[0106] Example 2

[0107] (1) Construction of the starting strain IBEWQ.

[0108] The starting strain IBEWQ was prepared from E. coli W3110 (competent cell products of which are available from several bioreagent companies) and was engineered to contain the trpE promoter at the tnaA site. S40F,M1293TThe DCBA gene was expressed in tandem, and AroF from E. coli K12 was inserted into the trpR site. P148L,Q152I,N8K , AorG L76V,P150L,D146N The tktA and ppsA genes were expressed in the tyrR site, the SerA gene from Bacillus subtilis was expressed in the tyrR site, and the promoters of the tyrA and pheA genes were simultaneously replaced with the PJ23114 promoter.

[0109] (2) The mutant strain IBEWQ-62 was obtained.

[0110] pKAP was electrotransformed into the IBEWQ strain to obtain IBEWQ-pKAP. This strain was used as the starting strain and serially subcultured into seed cultures containing different tryptophan concentrations (to which aminobenzyl resistance was added). After 12 h of incubation, the cultures were diluted 2-fold to detect biomass.

[0111] Table 1. Biomass detection values ​​at different tryptophan addition concentrations [Table 1]

[0112] The strains that showed increased growth rates in high-tryptophan culture medium were cultured in fermentation medium at 37°C, and one mutant strain that lost the pKAP plasmid and showed a significantly increased yield after plate purification was named IBEWQ-62.

[0113] Seed medium: 2.4 g / L K2HPO4, 9.6 g / L KH2PO4, 15 g / L yeast flour, 10 g / L rice bran, 5.0 g / L (NH4)2SO4, 1.0 g / L MgSO4·7H2O, 20 g / L glucose, natural pH.

[0114] Shake flask fermentation medium: 20 g / L glucose, 3.0 g / L yeast extract powder, 30 g / L rice bran, 1.6 g / L (NH4)2SO4, 2.0 g / L citric acid, 5.6 g / L K2HPO4, 2.0 g / L MgSO4·7H2O, 80 mg / L FeSO4·7H2O, 4.0 mg / L CoCl2·6H2O, 0.6 mg / L CuSO4·5H2O, 6.5 mg / L ZnSO4·7H2O, 20 mg / L Na2SO4, 4.5 mg / L MnSO4·H2O, 20 g / L CaCO3, pH 7.2.

[0115] Example 3 Analysis of tryptophan production by fermentation of mutant strains and genome sequence analysis

[0116] The starting strains IBEWQ and IBEWQ-62 were fermented. First, glycerol bacteria were activated in a slant medium, then transferred to a seed medium and cultured overnight. The resulting seeds were inoculated into 500 mL shake flasks containing 100 mL of fermentation medium and cultured at 220 rpm and 37°C. Samples were taken to detect the remaining glucose and tryptophan yield. After 45 hours of fermentation, the acid production level of IBEWQ-62 was significantly higher than that of the starting strain.

[0117] The IBEWQ-62 genome was extracted and whole genome sequence analysis was performed. Comparison of the genomes revealed that point mutations had occurred in the IBEWQ-62 genome compared to the starting strain, and the amino acid mutations are shown in Table 2.

[0118] Table 2. Mutations in the IBEWQ-62 genome compared to wild-type strains [Table 2-1] [Table 2-2]

[0119] The mutation sites of pepD, FadR and map are as follows:

[0120] For the mutant pepD gene, pepD S21T,G225A,A484K Three mutations, S21T, G225A, and A484K, respectively, were generated.

[0121] For the mutant FadR gene, FadR A140T,L171I and two mutations, A140T and L171I, respectively, occurred.

[0122] For the mutated map gene, map V94L,Q182N Two mutations, V94L and Q182N, were observed.

[0123] Example 4: Effect of mutant genes on fermentation efficiency

[0124] In the original strain, the promoters of genes (FadR, pepD, and map genes) that cause mutations in the amino acid sequence were replaced with promoters of different strengths, and the effects of each mutation on the valine fermentation yield were compared.

[0125] Using standard gene editing procedures, the promoters of the pepD, FadR, and map genes in the IBEWQ strain were replaced with the strong promoter PJ23119, and the pepD, FadR, and map promoters were replaced with the weak promoter PJ23114 using standard gene editing procedures. After construction, the correctly verified strains were incubated in seed medium for 12–16 h and then transferred to fermentation medium the following day.

[0126] After the fermentation was completed, the tryptophan production results of each production strain are shown in Table 3.

[0127] Table 3 [Table 3]

[0128] As can be seen from the fermentation results, the reduction of the fadR mutant gene contributes significantly to the increase in tryptophan yield, while the PepD mutant gene needs to be strengthened to be beneficial for tryptophan yield, and the map mutant does not affect tryptophan production.

[0129] Example 5 Construction of plasmids for overexpression or reduction of mutant genes

[0130] In strain IBEWQ-62, fadR A140T,L171I The promoter of the gene was replaced with PJ23114 and named IBEWQ-621. S21T,G225A,A484K The promoter of the gene was replaced with that of PJ23119, and the strain was named IBEWQ-622. A140T,L171I The knockout was named IBEWQ-623.

[0131] As shown in the figure, the acid production pattern clearly shows that enhanced expression of the pepD gene significantly improved the fermentation efficiency, while the reduction or complete knockout of fadR was optimal. Therefore, the composite strain IBEWQ-624 was constructed. In the IBEWQ-624 strain, fadR was knocked out in the genome and pepD was activated. S21T,G225A,A484K The promoter of the gene was replaced with PJ23119.

[0132] The fermentation level of IBEWQ-624 reached 4.91±0.28g / L, and the sugar-acid conversion rate was 24.5%. This indicates that the optimal solution for downregulating fadR is knockout of the fadR gene.

[0133] Example 6 Amplification and Verification

[0134] The starting strain IBEWQ and the mutant strain IBEWQ624 were inoculated into 500 mL shake flasks containing 100 mL of seed medium, and cultured at 37°C and 200 rpm for 12 to 16 hours. 600The values ​​were 11-13. The cultivated seed solution was inoculated into a 5L fermenter at a 10% (volume ratio) inoculum volume with an initial aeration ratio of 1.5 vvm and an initial rotation speed of 400 rpm. During fermentation, the pH was controlled at 7.0 by adding 25% aqueous ammonia, the fermentation temperature was controlled at 37±0.5°C, and the rotation speed and aeration were manually adjusted to maintain the dissolved oxygen at 20-30%. After approximately 6 hours of inoculation, the dissolved oxygen increased rapidly and the initial glucose was depleted. The automatic feed mode was activated, and 800 g / L of glucose was added to control the glucose concentration in the fermentation broth within 1 g / L.

[0135] After 16 hours of fermentation, samples were taken and measured every 2 to 4 hours. After 48 hours of fermentation, the tryptophan yield of the starting strain IBEWQ reached 42.12±4.61 g / L, with a sugar-acid conversion rate of 19.1%. The tryptophan yield of the mutant strain IBEWQ-624 reached 62.38±5.80 g / L, with a sugar-acid conversion rate of 24.1%.

[0136] Example 7 Use of the modified method with other strains

[0137] Starting from E. coli Nissle1917, the fadR gene was knocked out in the genome and the pepD gene was inserted into pepD S21T,G225A,A484K The promoter was then replaced with PJ23119 to obtain the recombinant strain N-RD. After 40 h of fermentation in a shake flask, 0.97 ± 0.06 g / L of tryptophan was detected in the fermentation broth of the starting strain, E. coli Nissle1917. The tryptophan yield in the fermentation broth of the recombinant strain reached 1.28 ± 0.03 g / L, a 1.32-fold increase.

[0138] Shake flask fermentation medium: 10 g / L glucose, 5.0 g / L yeast extract powder, 10 g / L rice bran, 6.0 g / L (NH4)2SO4, 3.0 g / L sodium citrate, 2.0 g / L L-glutamine, 1.0 g / L L-serine, 5.6 g / L K2HPO4, 3.0 g / L MgSO4·7H2O, 65 mg / L FeSO4·7H2O, 20 g / L calcium carbonate, pH 7.2.

[0139] Even when E. coli BL21, HB101, JM109, DH10B, or MG1655 was used as the starting strain, a 1.3- to 1.5-fold increase in yield was achieved.

[0140] The above are merely preferred specific embodiments of the present invention, and the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined based on the scope of protection of the claims.

Claims

1. An engineered bacterium, characterized in that it has been modified from a starting strain and contains a gene encoding at least one of a pepD mutant protein and a fadR mutant protein.

2. 2. The engineered fungus of claim 1, wherein the pepD mutant protein comprises at least one of the following mutations: S21T, G225A, and A484K.

3. 3. The engineered fungus of claim 1 or 2, characterized in that the pepD mutant protein comprises a sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:

1.

4. 2. The engineered fungus of claim 1, wherein the fadR mutant protein comprises at least one of the following mutations: A140T and L171I.

5. 5. The engineered fungus of claim 4, wherein the fadR mutant protein comprises a sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:

2.

6. 2. The engineered fungus of claim 1, wherein the pepD mutant protein has the amino acid sequence shown in SEQ ID NO: 1 and the fadR mutant protein has the amino acid sequence shown in SEQ ID NO:

2.

7. 2. The engineered fungus of claim 1, wherein the pepD mutant protein comprises the mutations S21T, G225A and A484, and the fadR mutant protein comprises the mutations A140T and L171I.

8. 2. The engineered fungus of claim 1, wherein the modification comprises increasing the expression level of the pepD protein of the starting strain.

9. 2. The engineered fungus of claim 1, wherein the modification comprises reducing the expression level of FadR protein in the starting strain.

10. 10. The engineered fungus according to claims 7 to 9, characterized in that the modification comprises knocking out the fadR gene in the genome of the engineered fungus containing a pepD mutant protein comprising mutations S21T, G225A and A484 and a fadR mutant protein comprising mutations A140T and L171I, and replacing the promoter of the gene encoding the pepD mutant protein in the genome of the starting strain with a strong promoter.

11. The engineered fungus according to any one of claims 1 to 10, wherein the starting strain is any one selected from Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, and yeast cells.

12. 12. The engineered fungus according to claims 1 to 11, characterized in that the starting strain is one selected from strain IBEWQ, mutant strain IBEWQ-62, mutant strain IBEWQ-624, E. coli Nissle 1917, E. coli BL21, E. coli HB101, E. coli JM109, E. coli DH10B or E. coli MG1655.

13. The engineered fungus according to claims 1 to 12, characterized in that the engineered fungus has an increased tryptophan yield under the same culture conditions compared to the unmodified starting strain.

14. 1. A method for constructing an engineered bacterium with a high tryptophan yield, comprising modifying at least one of a pepD protein and a fadR protein of a starting strain to obtain the engineered bacterium, wherein the modified engineered bacterium has a higher tryptophan yield than the starting strain under the same culture conditions.

15. the modification comprises mutating the pepD protein of the starting strain at an amino acid position, including at least one of the following mutations: S21T, G225A, and A484K, to generate a pepD mutant protein; or overexpressing the gene encoding the pepD protein or the pepD mutant protein using a high-copy plasmid as a vector; or replacing the promoter of the gene encoding the pepD protein or the pepD mutant protein in the genome of the starting strain with a strong promoter; or 15. The method of claim 14, comprising increasing the stability of mRNA transcribed from the gene for the pepD protein or the pepD mutant protein.

16. 16. The method of claim 15, wherein the alteration comprises causing the pepD mutant protein to contain a sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:

1.

17. The modification is: knocking out the gene encoding the fadR protein of the starting strain; or mutating the fadR protein of the starting strain at an amino acid site, including at least one of the A140T and L171I mutations, to generate a fadR mutant protein; or replacing the promoter of the gene encoding the FadR protein or the FadR mutant protein in the genome of the starting strain with a weak promoter; or 17. The method of claim 16, comprising suppressing the translation efficiency or reducing the stability of mRNA transcribed from the gene for the FadR protein or the FadR mutant protein.

18. 18. The method of claim 17, wherein the fadR mutant protein comprises a sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:

2.

19. 19. The method of claim 18, wherein the pepD mutant protein has the amino acid sequence shown in SEQ ID NO: 1 and the fadR mutant protein has the amino acid sequence shown in SEQ ID NO:

2.

20. The step of obtaining the engineered fungus comprises: culturing the starting strain in a culture medium containing different concentrations of tryptophan and detecting the biomass in the culture medium; determining a growth rate of the starting strain based on the biomass; and a step of fermenting the strain with an increased growth rate in a culture medium containing high tryptophan concentrations to obtain the engineered fungus capable of increasing tryptophan yield.

21. 21. The method of claim 20, wherein the high-concentration tryptophan has a concentration of 50 g / L to 70 g / L.

22. A mutant protein having a sequence that has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:1 or a sequence that has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:

2.

23. 23. The mutant protein of claim 22, comprising a pepD mutant protein, wherein the pepD mutant protein comprises at least one of the following mutations: S21T, G225A, and A484K.

24. 24. The mutant protein of claim 23, comprising a fadR mutant protein, wherein the pepD mutant protein comprises at least one of the following mutations: A140T and L171I.

25. A DNA molecule, characterized in that it comprises a gene encoding the mutant protein according to any one of claims 19 to 21.

26. A gene expression cassette, characterized in that it comprises the mutant protein according to any one of claims 19 to 21 or a gene encoding said mutant protein.

27. A recombinant vector, characterized in that it comprises the mutant protein according to any one of claims 19 to 21 or a gene encoding said mutant protein.

28. 10. Use of the engineered fungus of claim 1 in increasing tryptophan yield.

29. 29. The use according to claim 28, characterized in that it comprises fermenting the engineered fungus to obtain an increased yield of tryptophan.

30. The pepD mutant protein contained in the engineered bacterium is A protein (A1) whose amino acid sequence is SEQ ID NO: 1, or A protein (A2) having the same function as the amino acid sequence shown in SEQ ID NO: 1, in which one or more amino acid residues are substituted and / or deleted and / or added; or A protein (A3) having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity with the amino acid sequence defined by any one of (A1) to (A2) and having the same function; or The use according to claim 28, characterized in that it comprises any one of fusion proteins (A4) obtained by linking a tag to the N-terminus and / or C-terminus of a protein defined by any one of (A1) to (A3).

31. The use according to claim 28, characterized in that the fadR mutant protein contained in the engineered bacterium comprises any one of: a protein (A1) having the amino acid sequence of SEQ ID NO: 2; a protein (A2) having the same function as the amino acid sequence of SEQ ID NO: 2, with one or more amino acid residues substituted and / or deleted and / or added; a protein (A3) having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity with the amino acid sequence defined by any one of (A1) to (A2), with the same function; and a fusion protein (A4) obtained by linking a tag to the N-terminus and / or C-terminus of a protein defined by any one of (A1) to (A3).

32. The gene encoding the pepD mutant protein is pepD Ｓ２１Ｔ，Ｇ２２５Ａ，Ａ４８４Ｋ 29. The use according to claim 28, characterized in that it comprises a DNA molecule encoding a mutant protein and having 99% or more, 95% or more, 90% or more, 85% or more or 80% or more identity with the defined DNA sequence.

33. The gene encoding the fadR mutant protein is fadR Ａ１４０Ｔ，Ｌ１７１Ｉ 29. The use according to claim 28, characterized in that it comprises a DNA molecule encoding a mutant protein and having 99% or more, 95% or more, 90% or more, 85% or more or 80% or more identity with the defined DNA sequence.

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