Engineered bacterium, construction method therefor and use thereof

Abstract

An engineered bacterium for improving tryptophan yield, a construction method therefor, and a use thereof. A strain capable of tolerating a high concentration of tryptophan was obtained by screening. Genome sequencing and protein sequence analysis of the strain revealed that some proteins in the strain had undergone point mutations, and these mutants can improve the tryptophan yield. In order to further improve the tryptophan yield, modifications are made to a protein sequence expressed by a fadR gene or a protein sequence expressed by a pepD gene in a starting bacterium. Such modifications enable the obtained engineered bacterium to have a higher tryptophan yield than the starting bacterium. In the case of large-scale production, in a 5 L fermentation tank, the tryptophan yield can reach 62.38±5.80 g / L, and the sugar-to-acid conversion rate can reach 24.1%. Compared with the original bacterium, the tryptophan yield is increased by 1.48 folds, and the sugar-to-acid conversion rate is increased by 1.26 folds. The biological material and the use thereof relate to the technical field of molecular biology and have wide practical application value.

Description

An engineered bacterium and its construction method and application Cross-references

[0001] This application is based on and claims priority from Chinese application No. 202311807065.2 filed on December 26, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention relates to the technical field of molecular biology, and in particular to an engineered bacterium and a construction method and application thereof. Background Art

[0003] L-tryptophan is an essential amino acid widely used in food, feed, and medicine. The biosynthesis of tryptophan mainly includes three modules: the central carbon metabolism pathway (CCM), the shikimic acid (SHIK) pathway, and the chorismic acid (CHA) pathway. Its production pathway is long, requires many precursors, has strong feedback inhibition, and has relatively low production efficiency. The yield of industrial mature strains is difficult to meet market demand. With the continuous development of synthetic biology methods, more and more metabolic engineering strategies are being applied to construct cell factories for efficient L-tryptophan production. However, although the rational transformation of metabolic pathways has clear goals and significant effects, because microorganisms are complex systems, previous reports have shown that the expression of cytidine deaminase only slightly increases the mutation rate. The known metabolic network or transformation ideas cannot further achieve the goal of rapidly increasing production. Summary of the Invention

[0004] To address the above-mentioned issues, the present invention screened strains for tolerance and obtained a strain capable of withstanding high concentrations of tryptophan. After sequencing the strain's genome and analyzing its protein sequences, it was discovered that some proteins in the strain underwent point mutations, and these mutants were able to increase tryptophan production. The present invention induced mutations in the strain that were unrelated to the primary metabolic pathways for the synthesis of the target amino acid and the glycolytic pathway, resulting in a strain with superior properties. However, due to the high complexity of the metabolic network of an organism, the mutation results obtained in the present invention are subject to high uncertainty, making accurate prediction and regulation difficult in the early stages of research. Those skilled in the art typically do not consider whether modifications to these proteins would be beneficial for tryptophan production. Inspired by a mutation system involving the fusion of cytidine deaminase with T7 RNA polymerase, the applicants discovered that fusing cytidine deaminase with the α-subunit of Escherichia coli RNA polymerase could accelerate mutations to a level that supports effective adaptive evolution in E. coli without compromising cell viability. In industry, induced evolution is a means to rapidly improve the production capacity of strains. Combined with advanced technologies such as genome sequencing, it can combine improving production capacity with exploring principles, providing a basis and ideas for subsequent rational transformation, while improving the production level of strains and enhancing international competitiveness.

[0005] In one aspect, the present invention claims protection for an engineered bacterium with increased tryptophan production, obtained by modifying a starting bacterium. In some embodiments, the modified engineered bacterium 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 protection for a method for constructing an engineered bacterium with high tryptophan production. In some embodiments, the method comprises: modifying at least one of the pepD protein and the fadR protein of the starting bacterium to obtain the engineered bacterium; wherein, under identical culture conditions, the modified engineered bacterium produces a higher tryptophan yield than the starting bacterium.

[0006] In a third aspect, the present invention claims a biomaterial that can be used to increase tryptophan production.

[0007] In some embodiments, the biomaterial comprises a mutant protein having a sequence with at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 1 or a sequence with at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 2.

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

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

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

[0011] In a fourth aspect, the present invention claims protection for the use of an engineered bacterium in increasing tryptophan production.

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

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

[0014] The "pepD protein mutant" of the present invention is obtained by mutating the amino acid sequence set forth in SEQ ID NO:4. Specifically, the pepD protein mutant of the present invention comprises amino acid positions 21, 225, and 484 substituted with threonine, alanine, and lysine, respectively, corresponding to the amino acid sequence set forth in SEQ ID NO:4. Furthermore, the scope of the present invention also encompasses enzymes derived from Escherichia coli that have greater than 80%, preferably 90%, more preferably 95%, and most preferably 99% or greater homology to SEQ ID NO:1 and that function to degrade N-terminally unblocked dipeptides.

[0015] Similarly, the "fadR protein mutant" of the present invention is obtained by mutating the amino acid sequence set forth in SEQ ID NO:5. Specifically, the fadR protein mutant of the present invention comprises a threonine and isoleucine substitution at positions 140 and 171, respectively, corresponding to the amino acid sequence set forth in SEQ ID NO:5. Furthermore, the present invention also encompasses proteins derived from Escherichia coli that have greater than 80%, preferably 90%, more preferably 95%, and most preferably 99% or greater homology to SEQ ID NO:2 and possess dual DNA-binding transcriptional regulator activity.

[0016] As used herein, the term "exogenous" refers to the inclusion of substances not originally present in a system. For example, if a gene encoding an enzyme not originally present in a strain is introduced into the strain through transformation, such that the enzyme is expressed in the strain, then the enzyme is considered "exogenous" to the strain.

[0017] The term "enhancement" as used in this article not only includes effects higher than the original function due to the increase in the activity of the protein itself, but also can be carried out by at least one method selected from the following: 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 the chromosome with a sequence with strong activity, replacing the gene encoding the protein with a mutant gene to increase the activity of the protein, introducing modifications into the gene encoding the protein on the chromosome to enhance the activity of the protein, and can also include, without limitation, any existing method, as long as it can enhance the activity of the protein or enhance the activity of the introduced protein compared with the endogenous activity.

[0018] The term "introducing protein activity" as used herein has the meaning conventionally understood by those skilled in the art and can be implemented by methods known in the art, including but not limited to: inserting a polynucleotide comprising a polynucleotide sequence encoding a protein into a chromosome, and / or cloning the polynucleotide into a vector and introducing it into a microorganism, and / or directly increasing the copy number of the polynucleotide on the chromosome, and / or modifying a promoter having a polynucleotide encoding a protein to enhance the transcription initiation rate, and / or modifying the transcription of a polynucleotide encoding a protein to enhance its activity, and / or modifying the translation regulatory sequence of a messenger RNA carrying the polynucleotide encoding the protein to enhance the translation intensity, and / or modifying the polynucleotide encoding the protein itself to enhance mRNA stability, protein stability, relieve protein feedback inhibition, etc., and can also include, but is not limited to, any known method for introducing protein activity.

[0019] A vector is a DNA construct comprising a polynucleotide sequence encoding a target protein, operably linked to appropriate regulatory sequences to enable expression of the target protein in a host cell. After being introduced into a suitable host cell, the vector can replicate or function independently of the host cell genome, or can 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 bacteriophages. For example, pWE15, pET, and pUC vectors. In addition, by inserting the 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 the polynucleotide into the chromosome can be performed using any method known in the art, including, but not limited to, homologous recombination. Polynucleotides, including DNA and RNA encoding the target protein, can be inserted into the host cell chromosome in any form, as long as they are capable of expression in the host cell. This includes, but is not limited to, the polynucleotide can be introduced into the host cell in its native state and / or in the form of an expression cassette. An expression cassette is a genetic construct that includes all the necessary elements required for self-expression and can also be a self-replicating expression vector that can include a promoter operably bound to a polynucleotide, a transcription termination signal, a ribosome binding domain, and a translation termination signal.

[0020] Similarly, the term "weakening" as used herein refers to reducing, weakening, decreasing, or completely eliminating the activity of a protein, such as an enzyme. In specific embodiments, attenuating the activity of an enzyme can be achieved by partially or completely knocking out the enzyme's encoding gene, inactivating or partially inactivating the gene through mutation, altering the gene promoter or translational regulatory region to weaken its transcription or translation, altering the gene sequence to reduce the stability of its mRNA or destabilize the enzyme structure, regulating the gene through sRNA, or other methods or combinations thereof, including but not limited to the above methods.

[0021] The term "host cell" as used herein has the meaning commonly understood by those of ordinary skill in the art, i.e., a strain containing a protein or its protein mutant. In other words, the present invention can utilize any host cell, as long as the cell contains the target protein or its mutant and is capable of producing tryptophan. The host cell can be derived from preferably Escherichia coli (E.Coli). Specifically, the host of the present invention refers to a strain capable of producing tryptophan, that is, when the bacterium is cultured in culture, it can produce tryptophan and can accumulate tryptophan, or can secrete tryptophan into the culture medium, that is, it can obtain extracellular free tryptophan, and particularly refers to the ability to accumulate more tryptophan compared to the wild-type strain or the parent strain. In order to give the strain the ability to produce tryptophan, traditional breeding methods can be used, such as cultivating auxotrophic mutants, analogue-resistant strains, or metabolically controlled mutants capable of producing tryptophan, as well as methods for cultivating recombinant strains with improved activity of amino acid biosynthesis-related enzymes, or combinations of the above methods.

[0022] The term "containing the pepD and / or fadR protein mutants of the present invention" as used herein has the meaning conventionally understood by those skilled in the art and can be implemented by methods known in the art, including but not limited to: inserting a polynucleotide comprising a polynucleotide sequence encoding a protein into a chromosome, and / or cloning the polynucleotide into a vector and introducing it into a microorganism, and / or directly increasing copies of the polynucleotide on the chromosome, etc., and can also include, but is not limited to, any known method for introducing protein activity.

[0023] Those skilled in the art will appreciate that when mutating a wild-type polypeptide to enhance its activity, it is more important to identify sites that achieve the desired purpose. Therefore, based on the teachings of the present invention, those skilled in the art will substitute serine at position 21 with threonine, glycine at position 225 with alanine, and alanine at position 484 with lysine in the amino acid sequence of the pepD protein, and replace alanine at position 140 with threonine and leucine at position 171 with isoleucine in the amino acid sequence of the fadR protein, and test the relevant activities of the mutants.

[0024] Furthermore, it is readily apparent to those skilled in the art that altering a small number of amino acid residues in certain regions of a polypeptide, such as non-essential regions, will not substantially alter biological activity. For example, appropriate substitutions of certain amino acids will result in a sequence that does 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 are able to perform such substitutions and ensure that the resulting molecule still possesses the desired biological activity.

[0025] Therefore, it is readily apparent that further mutations can be made to the pepD and / or fadR proteins and mutants thereof of the present invention to yield further mutants that still possess the corresponding functions and activities. For example, those skilled in the art are well aware that adding or subtracting a number of amino acid residues, for example, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-3, and most preferably 1, at either end of a polypeptide will not affect the function of the resulting mutant. For example, to facilitate purification, technicians often attach a 6×His tag to either end of the resulting protein, and such a protein exhibits the same function as a protein without the 6×His tag. Therefore, the present invention encompasses conservative mutants derived from the present invention.

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

[0027] The present invention modifies the protein sequence expressed by the fadR gene or the pepD gene in a starting strain to produce engineered bacteria with increased tryptophan production compared to the starting strain. The starting strain is a tryptophan-producing strain. The fadR gene is modified to reduce its expression level or protein activity, or even to knock it out; the pepD gene is modified to mutate it and increase its expression level or protein activity. Under large-scale production conditions, tryptophan production in a 5L fermentor reached 62.38±5.80g / L, with a sugar-to-acid conversion rate of 24.1%. Compared to the starting strain, tryptophan production increased by 1.48 times, and the sugar-to-acid conversion rate increased by 1.26 times.

[0028] The deposit information of the engineered bacteria of the present invention is as follows.

[0029] Escherichia coli IBEWQ-624 was deposited in the China Center for Type Culture Collection on August 19, 2024, with the deposit number CCTCC NO: M 20241821. The deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 shows the tryptophan fermentation levels of the IBEWQ-62 strain and strains with varying degrees of weakening of fadR and / or strengthening of pepD. The results indicate that enhanced expression of the pepD gene significantly improves the fermentation effect, while the optimal solution for weakening fadR is to knock out the fadR gene. DETAILED DESCRIPTION

[0031] One of the embodiments of this specification provides an engineered bacterium, which is transformed from a starting bacterium and comprises a gene encoding at least one mutant protein of a pepD mutant protein and a fadR mutant protein.

[0032] In some embodiments, the pepD mutant protein comprises at least one mutation selected from the group consisting of S21T, G225A, and A484K. For example, the mutations involved in the pepD mutant protein may be S21T, G225A, or A484K mutations, each denoted as pepD S21T 、pepD G225A or pepD A484K For another example, the mutations involved in the pepD mutant protein may be S21T and G225A, G225A and A484K or S21T and A484K, respectively denoted as pepD S21T，G225A 、pepD G225A，A484K or pepD A484K，S21 .

[0033] In some embodiments, the mutations involved in the pepD mutant protein are S21T, G225A and A484K mutations, denoted as pepD A484K，S21，G225A .

[0034] Among them, pepD S21T The amino acid sequence of the pepD protein (SEQ ID NO: 4) shows that the serine at position 21 is replaced by threonine, and the relevant activities of the protein mutants are tested.

[0035] pepD G225A The amino acid sequence of the pepD protein was replaced by alanine at position 225 of glycine, and the activity of the mutant protein was tested.

[0036] pepD A484K The amino acid sequence of the pepD protein was replaced by lysine at position 484, and the activity of the mutant protein was tested.

[0037] In some embodiments, the pepD mutant protein comprises a sequence having at least 80% sequence identity to the amino acid sequence of 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 of 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 of 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 of 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 of 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 includes at least one of A140T and L171I mutations. For example, the mutation involved in the fadR mutant protein can be A140T or L171I mutation, denoted as fadR A140T or fadR L171I ; can also be A140T and L171I mutations, represented as fadR A140T，L171I .

[0040] Among them, fadR A140T The amino acid sequence of the fadR protein (SEQ ID NO: 5) was replaced by threonine at position 140, and the activity of the mutant protein was tested.

[0041] fadR L171I The amino acid sequence representing the fadR protein was replaced with isoleucine at position 171, and the activity of the mutant protein was tested.

[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 bacteria comprises a pepD mutant protein and a fadR mutant protein, wherein the pepD mutant protein comprises S21T, G225A, and A484 mutations, and the fadR mutant protein comprises A140T and L171I mutations.

[0044] In some embodiments, 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.

[0045] The engineered bacteria used in this article are modified from a starting strain. The starting strain refers to the original strain used for breeding. For example, the starting strain can be a spontaneously mutated strain during production, or it can be a strain with traits beneficial for further research or application, such as fast growth or low nutritional requirements. For another example, the starting strain can be a strain that has undergone other mutations or a mutator strain that is more sensitive to mutagens.

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

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

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

[0049] In some embodiments, the modification comprises attenuating the expression level of the FadR protein of the starting bacteria.

[0050] In some embodiments, the modification comprises knocking out the fadR gene in the genome of an engineered bacterium comprising a pepD mutant protein with S21T, G225A and A484 mutations and a fadR mutant protein with A140T and L171I mutations, and replacing the promoter of the gene encoding the pepD mutant protein in the genome of the starting bacterium with a strong promoter.

[0051] In the examples of this specification, the engineered bacteria after modification have increased tryptophan production compared to the unmodified starting bacteria under the same culture conditions.

[0052] The embodiments of this specification provide a method for constructing an engineered bacterium with high tryptophan production. In some embodiments, the method comprises: modifying at least one of the pepD protein and the fadR protein of a starting bacterium to obtain the engineered bacterium; wherein, under the same culture conditions, the modified engineered bacterium has a higher tryptophan production than the starting bacterium.

[0053] In some embodiments, the modification includes: mutating the amino acid sites in the pepD protein of the starting bacteria to generate a pepD mutant protein, wherein the mutation includes at least one of S21T, G225A and A484K mutations; 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 bacteria with a strong promoter; or improving the stability of the mRNA transcribed from the gene encoding 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 shown in SEQ ID NO:1.

[0055] In some embodiments, the modification comprises: knocking out the gene encoding the fadR protein in the starting bacteria; or mutating an amino acid position in the fadR protein of the starting bacteria to generate a fadR mutant protein, wherein the mutation comprises at least one of A140T and L171I mutations; or replacing the promoter of the gene encoding the FadR protein or the FadR mutant protein in the genome of the starting bacteria with a weak promoter; or inhibiting the translation efficiency or reducing the stability of mRNA transcribed from the gene encoding the FadR protein or the FadR mutant protein. 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.

[0056] In some embodiments, the modification comprises the following steps.

[0057] The endogenous pepD and / or fadR proteins of the starting bacteria are directly modified, or the exogenous pepD and / or fadR proteins are modified and then introduced into the starting bacteria; wherein the modification method is selected from at least one of the following technical means.

[0058] a. In the starting bacteria, the modification of the pepD protein may be any of the following.

[0059] (a1) Mutation of three amino acid sites in the pepD protein, namely S21T, G225A, and A484K, was performed to obtain the mutant protein pepD, hereinafter referred to as the mutant protein pepD S21T,G225A,A484K , whose amino acid sequence is shown in SEQ ID NO: 1.

[0060] (a2) Using high copy plasmid as vector, pepD protein or pepD S21T,G225A,A484K The gene encoding the mutant protein is overexpressed.

[0061] (a3) Transfecting genomic pepD protein or pepD S21T,G225A,A484K The promoter of the gene encoding the mutant protein is replaced with a stronger promoter.

[0062] (a4) increasing pepD protein or pepD S21T,G225A,A484K Stability of mRNA transcribed from genes encoding mutant proteins.

[0063] (a5) Any protein that can upregulate pepD protein or pepD S21T,G225A,A484K Methods and combinations thereof for mutating the expression level of a gene encoding a protein.

[0064] b. In the starting bacteria, the modification of the fadR protein may be any of the following.

[0065] (b1) Knock out the gene encoding the fadR protein in the starting bacteria.

[0066] (b2) Mutating two nucleotide positions of the gene encoding the fadR protein, A140T and L171I, to obtain a mutant protein of fadR, hereinafter referred to as mutant protein FadR A140T,L171I , whose amino acid sequence is shown in SEQ ID NO: 2.

[0067] (b3) FadR protein or FadR A140T,L171I The promoter of the gene encoding the mutant protein is replaced with a weak promoter.

[0068] (b4) Inhibition of FadR protein or FadR A140T,L171I The translation efficiency of the mRNA transcribed from the gene encoding the mutant protein is reduced or its stability is decreased.

[0069] (b5) Any method capable of weakening FadR protein or FadR A140T,L171I Methods and combinations thereof for mutating the expression level of a gene encoding a protein.

[0070] Furthermore, in a specific embodiment of the present invention, the starting bacteria is a tryptophan-producing strain; the starting bacteria is selected from any one of Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, yeast cells, etc.; when the starting bacteria is Escherichia coli, it is further preferably selected from strain IBEWQ, mutant strain IBEWQ-62, mutant strain IBEWQ-624, Escherichia coli Nissle1917, Escherichia coli BL21, Escherichia coli HB101, Escherichia coli JM109, Escherichia coli DH10B or Escherichia coli MG1655.

[0071] In some embodiments, the engineered bacteria is a mutant strain IBEWQ-624, which comprises a pepD mutant protein and a fadR mutant protein, 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.

[0072] In some embodiments, obtaining the engineered bacteria comprises: culturing the starting bacteria in a culture medium containing different concentrations of tryptophan, and detecting the biomass in the culture medium; determining the growth rate of the starting bacteria based on the biomass; and fermenting and culturing the strain with an accelerated growth rate in a high-concentration tryptophan culture medium to obtain the engineered bacteria capable of increasing tryptophan production.

[0073] In some embodiments, the high concentration of tryptophan has a concentration between 50 g / L and 70 g / L.

[0074] In a specific embodiment of the present invention, the engineered bacterium IBEWQ-624 is a strain in which the gene encoding the fadR protein is knocked out from the genome of the IBEWQ strain, and the gene encoding the pepD protein is mutated into pepD S21T,G225A,A484K At the same time as the protein coding gene, pepD S21T,G225A,A484K The promoter of the gene was replaced with the strong promoter PJ23119.

[0075] One of the embodiments of this specification provides a mutant protein, wherein the mutant protein has 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.

[0076] In some embodiments, the mutant protein comprises a pepD mutant protein comprising at least one mutation of S21T, G225A, and A484K.

[0077] In some embodiments, the mutant protein comprises a fadR mutant protein, and the pepD mutant protein comprises at least one mutation of A140T and L171I.

[0078] One of the embodiments of this specification provides a DNA molecule. In some embodiments, the DNA molecule comprises a gene encoding a mutant protein according to any one of claims 19 to 21.

[0079] One of the embodiments of this specification provides a gene expression cassette. In some embodiments, the gene expression cassette comprises the mutant protein as described above or a gene encoding the mutant protein.

[0080] One of the embodiments of this specification provides a recombinant vector. In some embodiments, the recombinant vector comprises the mutant protein as described above or a gene encoding the mutant protein.

[0081] One of the embodiments of this specification provides an application of an engineered bacterium in increasing tryptophan production.

[0082] In some embodiments, the use comprises fermentation culturing the engineered bacteria to obtain tryptophan with increased yield.

[0083] In some embodiments, the pepD mutant protein contained in the engineered bacteria includes any of the following proteins: (A1) a protein having an amino acid sequence of SEQ ID NO: 1; or (A2) a protein having the same function by replacing and / or deleting and / or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 1; or (A3) a protein having more than 99%, more than 95%, more than 90%, more than 85% or more homology with the amino acid sequence defined in any one of (A1)-(A2) and having the same function; or (A4) a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of any one of the proteins defined in (A1)-(A3).

[0084] In some embodiments: the fadR mutant protein contained in the engineered bacteria includes any of the following proteins: (A1) a protein having an amino acid sequence of SEQ ID NO: 2; (A2) a protein having the same function as the amino acid sequence shown in SEQ ID NO: 2 by substitution and / or deletion and / or addition of one or more amino acid residues; (A3) a protein having more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology with the amino acid sequence defined in any one of (A1)-(A2) and having the same function; (A4) a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of any one of the proteins defined in (A1)-(A3).

[0085] In some embodiments, the gene encoding the pepD mutant protein includes a gene encoding pepD S21T,G225A,A484K The DNA sequence defined by the mutant protein has a DNA molecule with a homology of more than 99%, more than 95%, more than 90%, more than 85% or more than 80%.

[0086] In some embodiments, the gene encoding the fadR mutant protein includes a gene encoding fadR A140T,L171I The DNA sequence defined by the mutant protein has a DNA molecule with a homology of more than 99%, more than 95%, more than 90%, more than 85% or more than 80%.

[0087] The present invention claims any one of the following biological materials, which can be used to increase tryptophan production.

[0088] (I) Protein: pepD S21T,G225A,A484K Mutant protein and / or fadR A140T,L171I Mutant protein.

[0089] (II) Gene: pepD S21T,G225A,A484K Mutant protein and / or fadR A140T,L171I The gene encoding the mutant protein.

[0090] (III) Expression cassette: containing the pepD S21T,G225A,A484K Mutant protein and / or fadR A140T,L171I An expression cassette of the gene encoding the mutant protein or an expression cassette containing the DNA fragment.

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

[0092] (V) Recombinant bacteria: containing the 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 DNA fragment.

[0093] (a) Application of biomaterials in increasing tryptophan production in starter cultures.

[0094] (b) Application of biomaterials in the production of tryptophan.

[0095] (c)pepD S21T,G225A,A484K Mutant protein and / or fadR A140T,L171I Application of mutant proteins in increasing tryptophan production in starting bacteria.

[0096] (d)pepD S21T,G225A,A484K Mutant protein and / or fadR A140T,L171I Application of mutant proteins in the production of tryptophan.

[0097] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

[0098] In the present invention, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used can be purchased from commercial channels. Example 1: Screening for tolerant strains with accelerated growth in tryptophan fermentation broth

[0099] Fusion of cytidine deaminase to the α-subunit of Escherichia coli RNA polymerase can accelerate mutations to a level that supports effective adaptive evolution of E. coli without reducing cell viability.

[0100] The gene sequences of the α-subunit of RNA polymerase and cytidine deaminase in the genome of the starting strain were amplified using primers RNAPα-F / R and CDA-F / R. The two end sequences were then fused and expressed using RNAPα-F and CDA-R as primers. After double digestion with EcoRI and NcoI, the fusion was ligated into the temperature-sensitive plasmid pKD46 (GenBank accession no.: MF287367), which was also double digested with EcoRI. The constructed plasmid, named pKAP, contains an arabinose-inducible promoter that regulates the expression of the α-subunit of RNA polymerase and cytidine deaminase genes.

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

[0102] RNAPα-F: GAATTCatgcagggttctgtgacag; SEQ ID NO:7.

[0103] RNAPα-R: CGATCCGCCACCGCCAGAGCCACCTCCGCCctcgtcagcgatgcttgccggtg; SEQ ID NO: 8.

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

[0105] CDA-R: CCATGGttaagcgagaagcactcgg; SEQ ID NO: 10. Example 2

[0106] (1) Construction of the starting strain IBEWQ.

[0107] The starting strain IBEWQ is based on Escherichia coli W3110 (competent cell products can be purchased from many biological reagent companies) and expresses trpE in tandem with the tac promoter at the tnaA site. S40F,M1293T DCBA gene, expressing AroF from Escherichia coli K12 at the trpR site P148L,Q152I,N8K , AorG L76V,P150L,D146N , tktA and ppsA genes, and expressed the SerA gene from Bacillus subtilis at the tyrR site; at the same time, the promoters of the tyrA and pheA genes were replaced with the PJ23114 promoter.

[0108] (2) Obtaining the mutant strain IBEWQ-62:

[0109] pKAP was electroporated into the IBEWQ strain to obtain IBEWQ-pKAP. Using this strain as the starting strain, it was serially subcultured and evolved in seed culture medium containing different tryptophan concentrations (with ampicillin added for resistance). After 12 hours of culture, the biomass was measured by diluting the culture 2-fold. Table 1 Biomass values ​​at different tryptophan concentrations.

[0110] The strains that grew faster in the high-concentration tryptophan culture medium were cultured at 37°C in a fermentation medium and lost the plasmid pKAP. After plate purification, one mutant strain with significantly improved yield was named IBEWQ-62.

[0111] Seed culture medium: 2.4 g / L K2HPO4, 9.6 g / L KH2PO4, 15 g / L yeast powder, 10 g / L rice bran, 5.0 g / L (NH4)2SO4, 1.0 g / L MgSO4·7H2O, 20 g / L glucose, natural pH.

[0112] Shake flask fermentation medium: 20 g / L glucose, 3.0 g / L yeast extract, 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 calcium carbonate, pH 7.2. Example 3: Fermentation and genome sequencing analysis of mutant strains for tryptophan production

[0113] Fermentation was performed on the starting strains IBEWQ and IBEWQ-62. First, the glycerol strain was activated on a slant medium and then transferred to seed culture medium to culture IBEWQ and IBEWQ-62 overnight. The resulting seed was inoculated into a 500mL shake flask containing 100mL of fermentation medium. The culture was incubated at 220rpm and 37°C. Samples were collected to measure residual glucose and tryptophan production. After 45 hours of fermentation, IBEWQ-62 produced significantly higher acid levels than the starting strain.

[0114] The IBEWQ-62 genome was extracted and sequenced. Comparative genome analysis revealed that the IBEWQ-62 genome had point mutations compared to the starting strain, of which the amino acid mutations are shown in Table 2. Table 2 Mutations in the IBEWQ-62 genome compared to wild strains

[0115] The mutation sites of pepD, FadR, and map are as follows.

[0116] For the mutated pepD gene, it is denoted as pepD S21T,G225A,A484K , three mutations occurred, namely S21T, G225A and A484K.

[0117] For the mutated FadR gene, it is denoted as FadR A140T,L171I , two mutations occurred, namely A140T and L171I.

[0118] The mutated map gene is denoted as mapV94L, Q182N, and two mutations, V94L and Q182N, have occurred. Example 4: Effect of the mutated gene on the fermentation effect

[0119] The promoters of the amino acid sequence mutant genes (FadR, pepD and map gene) were replaced with different strengths in the original strain, and the effects of various mutants on the valine fermentation yield were compared.

[0120] Following standard gene editing procedures, the promoters of the pepD, FadR, and map genes were replaced with the strong promoter PJ23119 in the IBEWQ strain. Following standard gene editing procedures, the promoters of the pepD, FadR, and map genes were replaced with the weak promoter PJ23114. After construction, verified correct strains were activated in seed culture for 12-16 hours and then inoculated into fermentation culture the next day.

[0121] After the fermentation was completed, the tryptophan production results of each production strain were shown in Table 3. Table 3

[0122] From the fermentation results, it can be seen that weakening the fadR mutant gene is very helpful for increasing tryptophan production, while the PepD mutant gene needs to be strengthened to be beneficial to tryptophan production, and the map mutant has no effect on tryptophan production. Example 5. Construction of plasmids for overexpression or weakening of mutant genes

[0123] In the IBEWQ-62 strain, fadR A140T,L171I The promoter of the gene was replaced with PJ23114 and designated as IBEWQ-621;

[0124] pepD S21T,G225A,A484K The promoter of the gene was replaced with PJ23119 and designated as IBEWQ-622;

[0125] Knockout of fadR in the IBEWQ-62 genome A140T,L171I, recorded as IBEWQ-623.

[0126] The acid production is shown in the figure. It can be seen that the enhanced expression of the pepD gene significantly improves the fermentation effect, and the weakening or complete knockout of fadR is the best. Thus, the combined strain IBEWQ-624 was constructed. The IBEWQ-624 is a strain IBEWQ-62, in which fadR is knocked out on the genome and pepD is replaced. S21T,G225A,A484K The promoter of the gene was replaced with PJ23119.

[0127] The fermentation level of IBEWQ-624 reached 4.91±0.28g / L, and the sugar-acid conversion rate was 24.5%. This shows that the best solution for weakening the regulation of fadR is to knock out the fadR gene. Example 6: Amplification Verification

[0128] The starting strain IBEWQ and the mutant strain IBEWQ624 were inoculated into 500 mL shake flasks containing 100 mL seed culture medium, and cultured at 37 ° C and 200 rpm for 12-16 h. 600 The value is 11-13. The cultured seed liquid was inoculated into a 5L fermenter at a volume ratio of 10% with an initial ventilation ratio of 1.5 vvm and an initial rotation speed of 400 rpm. During the fermentation process, 25% ammonia water was added to control the pH to 7.0; the fermentation temperature was controlled at 37±0.5°C; the rotation speed and ventilation were manually adjusted to maintain the dissolved oxygen at 20%-30%. About 6 hours after inoculation, the dissolved oxygen suddenly increased and the initial glucose was exhausted. The automatic feeding mode was activated and the glucose concentration in the fermentation liquid was controlled within 1g / L by adding 800g / L glucose.

[0129] After 16 hours of fermentation, samples were taken every 2 to 4 hours for measurement. After 48 hours of fermentation, the tryptophan production of the starting strain IBEWQ reached 42.12 ± 4.61 g / L, and the sugar-acid conversion rate was 19.1%. The tryptophan production of the mutant strain IBEWQ-624 reached 62.38 ± 5.80 g / L, and the sugar-acid conversion rate was 24.1%. Example 7: Application of the modified method in other strains

[0130] Using Escherichia coli Nissle1917 as the starting strain, the fadR gene was knocked out in its genome, and the pepD gene was mutated into pepD S21T,G225A,A484K The promoter was replaced with PJ23119, resulting in the recombinant strain N-RD. After 40 hours of shake flask fermentation, 0.97±0.06 g / L of tryptophan was detected in the fermentation broth of the starting strain, E. coli Nissle1917. The tryptophan production in the fermentation broth of the recombinant strain reached 1.28±0.03 g / L, a 1.32-fold increase.

[0131] 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.

[0132] Using E. coli BL21, HB101, JM109, DH10B, and MG1655 as the starting strains can also achieve a 1.3-1.5-fold increase in yield.

[0133] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes 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 in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. An engineered bacterium, characterized in that, The engineered bacterium is modified from a parental bacterium and contains a gene encoding at least one mutant protein among a pepD mutant protein and a fadR mutant protein.

2. The engineered bacterium according to claim 1, wherein The pepD mutant protein includes at least one mutation among S21T, G225A, and A484K.

3. The engineered bacterium according to any one of claims 1-2, characterized in that, The pepD mutant protein contains a sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:

1.

4. The engineered bacterium according to claim 1, characterized in that, The fadR mutant protein includes at least one mutation among A140T and L171I.

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

2.

6. The engineered bacterium according to 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. The engineered bacterium according to claim 1, wherein The pepD mutant protein includes S21T, G225A, and A484 mutations, and the fadR mutant protein includes A140T and L171I mutations.

8. The engineered bacterium according to claim 1, wherein The modification includes enhancing the expression level of the pepD protein of the parental bacterium.

9. The engineered bacterium according to claim 1, wherein The modification includes weakening the expression level of the FadR protein of the parental bacterium.

10. The engineered bacteria according to claims 7-9, characterized in that, The modification includes knocking out the fadR gene on the genome of an engineered bacterium containing a pepD mutant protein with S21T, G225A, and A484 mutations and a fadR mutant protein with A140T and L171I mutations, and replacing the promoter of the gene encoding the pepD mutant protein in the genome of the parental bacterium with a strong promoter.

11. The engineered bacterium according to any one of claims 1-10, characterized in that, The parental bacterium is selected from any one of Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, and yeast cells.

12. The engineered bacteria according to claims 1-11, characterized in that, The parental bacterium is selected from one of strain IBEWQ, mutant strain IBEWQ-62, mutant strain IBEWQ-624, Escherichia coli Nissle1917, Escherichia coli BL21, Escherichia coli HB101, Escherichia coli JM109, Escherichia coli DH10B, or Escherichia coli MG1655.

13. The engineered bacterium according to any one of claims 1-12, characterized in that, Compared with the unmodified parental bacterium, under the same culture conditions, the tryptophan yield of the modified engineered bacterium increases.

14. A method for constructing an engineered bacterium with high tryptophan production, characterized in that, The method includes: modifying at least one of the pepD protein and the fadR protein of a parental bacterium to obtain the engineered bacterium; wherein, under the same culture conditions, the modified engineered bacterium has a higher tryptophan yield than the parental bacterium.

15. The method according to claim 14, wherein The modification includes: mutating amino acid sites in the pepD protein of the parental bacterium to generate a pepD mutant protein, and the mutation includes at least one of S21T, G225A, and A484K mutations; or Using a high-copy plasmid as a vector to overexpress the gene encoding the pepD protein or the pepD mutant protein; or Replacing the promoter of the gene encoding the pepD protein or the pepD mutant protein in the genome of the parental bacterium with a strong promoter; or Enhancing the stability of the mRNA transcribed from the gene of the pepD protein or the pepD mutant protein.

16. The method according to claim 15, wherein The modification includes: the pepD mutant protein contains a sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:

1.

17. The method according to claim 16, wherein The modification includes: knocking out the gene encoding the fadR protein in the starting bacterium; or mutating the amino acid sites in the fadR protein of the starting bacterium to generate a fadR mutant protein, the mutation including at least one of A140T and L171I mutations; or replacing the promoter of the gene encoding the FadR protein or the FadR mutant protein in the genome of the starting bacterium with a weak promoter; or inhibiting the translation efficiency of the mRNA transcribed from the gene of the FadR protein or the FadR mutant protein or reducing its stability.

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

2.

19. The method according to claim 18, characterized in that, characterized in that, 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 method according to any one of claims 14-19, characterized in that, Obtaining the engineered bacterium includes: culturing the starting bacterium in a culture medium containing different concentrations of tryptophan, and detecting the biomass in the culture medium; determining the growth rate of the starting bacterium based on the biomass; and fermenting and culturing the strain with an accelerated growth rate in a high-concentration tryptophan culture medium to obtain the engineered bacterium capable of increasing the tryptophan yield.

21. The method according to claim 20, wherein The concentration of the high-concentration tryptophan is between 50 g / L and 70 g / L.

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

2.

23. The mutant protein according to claim 22, characterized in that, The mutant protein includes a pepD mutant protein, and the pepD mutant protein includes at least one mutation among S21T, G225A, and A484K.

24. The mutant protein according to claim 23, wherein, The mutant protein includes a fadR mutant protein, and the pepD mutant protein includes at least one of A140T and L171I mutations.

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

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

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

28. Use of the engineered bacterium according to claim 1 in increasing the tryptophan yield.

29. The application according to claim 28, characterized in that, The use includes fermenting and culturing the engineered bacterium to obtain tryptophan with increased yield.

30. The application according to claim 28, characterized in that, The pepD mutant protein contained in the engineered bacterium includes any of the following proteins: (A1) a protein with the amino acid sequence of SEQ ID NO:1; or (A2) A protein having the amino acid sequence shown in SEQ ID NO: 1 with substitution and / or deletion and / or addition of one or several amino acid residues and having the same function; or (A3) A protein having a homology of more than 99%, more than 95%, more than 90%, more than 85% or more than 80% with the amino acid sequence defined in any of (A1)-(A2) and having the same function; or (A4) A fusion protein obtained by linking a tag to the N-terminus and / or C-terminus of the protein defined in any of (A1)-(A3).

31. The application according to claim 28, characterized in that: The fadR mutant protein contained in the engineered bacterium includes any of the following proteins: (A1) A protein having the amino acid sequence of SEQ ID NO: 2; (A2) A protein having the amino acid sequence shown in SEQ ID NO: 2 with substitution and / or deletion and / or addition of one or several amino acid residues and having the same function; (A3) A protein having a homology of more than 99%, more than 95%, more than 90%, more than 85% or more than 80% with the amino acid sequence defined in any of (A1)-(A2) and having the same function; (A4) A fusion protein obtained by linking a tag to the N-terminus and / or C-terminus of the protein defined in any of (A1)-(A3).

32. The application according to claim 28, characterized in that, The gene encoding the pepD mutant protein includes a DNA molecule having a homology of more than 99%, more than 95%, more than 90%, more than 85% or more than 80% with the DNA sequence defined by the encoding pepD S21T,G225A,A484K mutant protein.

33. The application according to claim 28, wherein The gene encoding the fadR mutant protein includes a DNA molecule having a homology of more than 99%, more than 95%, more than 90%, more than 85% or more than 80% with the DNA sequence defined by the encoded fadR A140T,L171I mutant protein.

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