ENGINEERED STRAIN AND THE CONSTRUCTION METHOD AND USE THEREOF

Abstract

An engineered strain for improving tryptophan production and a construction method and use thereof are provided. By screening out a strain capable of tolerating high-concentration tryptophan and performing genomic sequencing and protein sequence analysis on the strain, it is found that certain proteins in the strain undergo point mutations and these mutations are capable of enhancing tryptophan production. To increase tryptophan production, protein sequences encoded by fadR or pepD genes in a parent strain are modified. These modifications result in an engineered strain with significantly higher tryptophan production compared to the parent strain. Under scaled-up production conditions, the tryptophan production reaches 62.385.80 g/L in a 5 L fermenter, with a glucose-to-tryptophan yield of 24.1%. Compared to an original strain, tryptophan production increases by 1.48-fold, and the glucose-to-tryptophan yield improves by 1.26-fold. The biological materials and its use belong to the technical field of molecular biology and possess broad practical application value.

Claims

1. An engineered strain, wherein the engineered strain comprises a gene encoding at least one of a pepD mutant protein or a fadR mutant protein.

2. The engineered strain of claim 1, wherein the pepD mutant protein includes at least one of mutations S21T, G225A, and A484K.

3. The engineered strain of claim 1, wherein the pepD mutant protein includes a sequence having at least 80% sequence identity to an amino acid sequence shown in SEQ ID NO: 1.

4. The engineered strain of claim 1, wherein the fadR mutant protein includes at least one of mutations A140T and L171I.

5. The engineered strain of claim 4, wherein the fadR mutant protein includes a sequence having at least 80% sequence identity to an amino acid sequence shown in SEQ ID NO: 2.

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

7. The engineered strain of claim 1, wherein the pepD mutant protein includes mutations S21T, G225A, and A484K, and the fadR mutant protein includes mutations A140T and L171I.

8. The engineered strain of claim 1, wherein the engineered strain is obtained by modification from a parent strain, and the modification includes enhancing an expression level of a pepD protein of the parent strain.

9. The engineered strain of claim 8, wherein the modification includes weakening an expression level of a fadR protein of the parent strain.

10. The engineered strain of claim 8, wherein the modification includes knocking out a fadR gene in a genome of the engineered strain including the pepD mutant protein with mutations S21T, G225A, and A484K and the fadR mutant protein with mutations A140T and L171I, and replacing a promoter of a gene encoding the pepD mutant protein in a genome of the parent strain with a strong promoter.

11. The engineered strain of claim 8, wherein the parent strain is selected from any one of Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, or yeast cells.

12. The engineered strain of claim 8, wherein the parent strain is selected from one of a strain IBEWQ, a mutant strain IBEWQ-62, a mutant strain IBEWQ-624, an Escherichia coli Nissle1917, Escherichia coli BL21, Escherichia coli HB101, Escherichia coli JM109, Escherichia coli DH10B, or Escherichia coli MG1655.

13. The engineered strain of claim 8, wherein under a same culture condition, a tryptophan production of the engineered strain is increased compared with the parent strain.

14. A method for constructing an engineered strain with a high tryptophan production, comprising: obtaining the engineered strain by modifying at least one of a pepD protein or a fadR protein of a parent strain; wherein under a same culture condition, a tryptophan production of the engineered strain is higher than a tryptophan production of the parent strain.

15. The method of claim 14, wherein the modification includes: generating a pepD mutant protein by introducing a mutation at an amino acid site of the pepD protein of the parent strain, wherein the mutation includes at least one of mutations S21T, G225A, and A484K; or overexpressing a gene encoding the pepD protein or the pepD mutant protein using a high-copy plasmid as a vector; or replacing a promoter of a gene encoding the pepD protein or the pepD mutant protein in a genome of the parent strain with a strong promoter; or improving stability of mRNA transcribed from the gene encoding the pepD protein or the pepD mutant protein.

16. The method of claim 15, wherein the modification includes: the pepD mutant protein including a sequence having at least 80% sequence identity to an amino acid sequence shown in SEQ ID NO: 1.

17. The method of claim 14, wherein the modification includes: knocking out a gene encoding the fadR protein of the parent strain; or generating a fadR mutant protein by introducing a mutation at an amino acid site of the fadR protein of the parent strain, wherein the mutation includes at least one of mutations A140T and L1711; or replacing a promoter of a gene encoding the fadR protein or the fadR mutant protein in a genome of the parent strain with a weak promoter; or inhibiting translation efficiency or reducing stability of mRNA transcribed from the gene encoding the fadR protein or the fadR mutant protein.

18. The method of claim 17, wherein the fadR mutant protein includes a sequence having at least 80% sequence identity to an amino acid sequence shown in SEQ ID NO: 2.

19. The method of claim 18, wherein the pepD mutant protein has an amino acid sequence shown in SEQ ID NO: 1, and the fadR mutant protein has an amino acid sequence shown in SEQ ID NO: 2.

20. The method of claim 14, wherein the obtaining the engineered strain includes: culturing the parent strain in culture media including different concentrations of tryptophan and measuring a biomass in each culture medium; determining a growth rate of the parent strain based on the biomass; and obtaining the engineered strain capable of increasing a tryptophan production by fermenting a strain with a faster growth rate in a culture medium including high-concentration tryptophan.

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

22. A mutant protein, wherein the mutant protein includes a sequence having at least 80% sequence identity to an amino acid sequence shown in SEQ ID NO: 1 or a sequence having at least 80% sequence identity to an amino acid sequence shown in SEQ ID NO: 2.

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

24. The mutant protein of claim 22, wherein the mutant protein includes a fadR mutant protein, wherein the fadR mutant protein includes at least one of mutations A140T and L171I.

25. A DNA molecule, comprising a gene encoding the mutant protein of claim 19.

26. A gene expression cassette, comprising the mutant protein of claim 19 or a gene encoding the mutant protein.

27. A recombinant vector, comprising the mutant protein of claim 19 or a gene encoding the mutant protein.

28-29. (canceled)

30. The engineered strain of claim 1, wherein the pepD mutant protein included in the engineered strain includes any one of the following: (A1) a protein having an amino acid sequence shown in SEQ ID NO: 1; or (A2) a protein derived from the amino acid sequence shown in SEQ ID NO: 1 containing one or more amino acid substitutions, deletions, and/or insertions, and possessing the same function as the protein having an amino acid sequence shown in SEQ ID NO: 1; or (A3) a protein having an amino acid sequence with at least 90%, homology to any one of the amino acid sequences defined in (A1) and (A2), and possessing the same function as the proteins defined in (A1) and (A2); or (A4) a fusion protein obtained by linking a tag to the N-terminus and/or C-terminus of any one of the proteins defined in (A1) to (A3).

31. The engineered strain of claim 1, wherein the fadR mutant protein included in the engineered strain includes any one of the following: (A1) a protein having an amino acid sequence shown in SEQ ID NO: 2; or (A2) a protein derived from the amino acid sequence shown in SEQ ID NO: 2 containing one or more amino acid substitutions, deletions, and/or insertions, and possessing the same function as the protein having an amino acid sequence shown in SEQ ID NO: 2; or (A3) a protein having an amino acid sequence with at least 90%, homology to any one of the amino acid sequences defined in (A1) and (A2), and possessing the same function as the proteins defined in (A1) and (A2); or (A4) a fusion protein obtained by linking a tag to the N-terminus and/or C-terminus of any one of the proteins defined in (A1) to (A3).

32. The engineered strain of claim 1, wherein the gene encoding the pepD mutant protein includes a DNA molecule having at least 90% homology to a DNA sequence defined by a gene encoding the pepD.sup.S21T, G225A, A484K mutant protein.

33. The engineered strain of claim 1, wherein the gene encoding the fadR mutant protein includes a DNA molecule having at least 90% homology to a DNA sequence defined by a gene encoding the fadR.sup.A140T, L171I mutant protein.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG. 1 is a schematic diagram illustrating tryptophan fermentation levels of a strain IBEWQ-62 and strains derived from the strain IBEWQ-62 with different weakening degrees of a fadR gene or/and with different enhancement degrees of a pepD gene in a genome. The diagram indicates the enhanced expression of the pepD gene significantly improves the fermentation performance while weakening or even completely knocking out the fadR gene yields the best improvement in fermentation performance.

DETAILED DESCRIPTION

[0032] One of embodiments of the present disclosure provides an engineered strain, the engineered strain is obtained by modification from a parent strain, and the engineered strain comprises a gene encoding at least one of a pepD mutant protein or a fadR mutant protein.

[0033] In some embodiments, the pepD mutant protein includes at least one of mutations S21T, G225A, and A484K. For example, a mutation involved in the pepD mutant protein may be mutations S21T, G225A, or A484K, denoted as pepD.sup.S21T, pepD.sup.G225A, or pepD.sup.A484K, respectively. As another example, the mutation involved in the pepD mutant protein may be the mutations S21T and G225A, mutations G225A and A484K, or mutations S21T and A484K, denoted as pepD.sup.S21T, G225A, pepD.sup.G225A, A484K or pepD.sup.A484K, S21T, respectively.

[0034] In some embodiments, the mutation involved in the pepD mutant protein may be mutations S21T, G225A, and A484K, denoted as pepD.sup.A484K, S21T, G225A.

[0035] The pepD.sup.S21T refers to the serine at position 21 of an amino acid sequence (SEQ ID NO: 4) of the pepD protein being substituted by threonine, and the relevant activity of this protein mutant is then tested.

[0036] The pepD.sup.G225A refers to the glycine at position 225 of the amino acid sequence of the pepD protein being substituted by alanine, and the relevant activity of this protein mutant is then tested.

[0037] The pepD.sup.A484K refers to the alanine at position 484 of the amino acid sequence of the pepD protein being substituted by lysine, and the relevant activity of this protein mutant is then tested.

[0038] In some embodiments, the pepD mutant protein includes a sequence having at least 80% sequence identity to an amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the pepD mutant protein includes a sequence having at least 85% sequence identity to the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the pepD mutant protein includes a sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the pepD mutant protein includes a sequence having at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the pepD mutant protein includes a sequence having at least 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 1.

[0039] In some embodiments, the pepD mutant protein has an amino acid sequence shown in SEQ ID NO: 1.

[0040] In some embodiments, the fadR mutant protein includes at least one of mutations A140T and L171I. For example, a mutation involved in the fadR mutant protein may be a mutation A140T or a mutation L171I, denoted as fadR.sup.A140T or fadR.sup.L171I, respectively; or the mutation involved in the fadR mutant protein may be mutations A140T and L171I, denoted as fadR.sup.A140T, L171I.

[0041] The fadR.sup.A140T refers to the alanine at position 140 of an amino acid sequence (SEQ ID NO: 5) of the fadR protein being substituted by threonine, and the relevant activity of this protein mutant is tested.

[0042] The fadR.sup.L171I refers to the leucine at position 171 of the amino acid sequence of the fadR protein being substituted by isoleucine, and the relevant activity of this protein mutant is tested.

[0043] In some embodiments, the fadR mutant protein includes a sequence having at least 80% sequence identity to an amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the fadR mutant protein includes a sequence having at least 85% sequence identity to the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the fadR mutant protein includes a sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the fadR mutant protein includes a sequence having at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the fadR mutant protein includes a sequence having at least 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 2.

[0044] In some embodiments, the engineered strain comprises the pepD mutant protein and the fadR mutant protein. The pepD mutant protein includes mutations S21T, G225A, and A484K, and the fadR mutant protein includes mutations A140T and L171I.

[0045] In some embodiments, the pepD mutant protein has an amino acid sequence shown in SEQ ID NO: 1, and the fadR mutant protein has an amino acid sequence shown in SEQ ID NO: 2.

[0046] The engineered strain in the present disclosure is obtained by modification from the parent strain. The parent strain refers to an original strain used for breeding. For example, the parent strain may originate from a spontaneously mutated strain in production, or from a strain exhibiting traits favorable for further research or application, such as rapid growth or low nutritional requirements. For example, the parent strain may be a strain that has already undergone other mutations or a mutator variant with high sensitivity to mutagens.

[0047] In some embodiments, the parent strain is selected from any one of Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, or yeast cells.

[0048] In some embodiments, the parent strain is selected from one of a strain IBEWQ, a mutant strain IBEWQ-62, a mutant strain IBEWQ-624, an Escherichia coli Nissle 1917, Escherichia coli BL21, Escherichia coli HB101, Escherichia coli JM109, Escherichia coli DH10B, or Escherichia coli MG1655.

[0049] In some embodiments, the modification includes enhancing an expression level of a pepD protein of the parent strain.

[0050] In some embodiments, the modification includes weakening an expression level of a fadR protein of the parent strain.

[0051] In some embodiments, the modification includes knocking out a fadR gene in a genome of the engineered strain including the pepD mutant protein with mutations S21T, G225A, and A484K and the fadR mutant protein with mutations A140T and L171I, and replacing the promoter of a gene encoding the pepD mutant protein in a genome of the parent strain with a strong promoter.

[0052] Under a same culture condition, a tryptophan production of the engineered strain is increased compared with the parent strain.

[0053] Embodiments of the present disclosure provide a method for constructing an engineered strain. In some embodiments, the method comprises: obtaining the engineered strain by modifying at least one of a pepD protein or a fadR protein of the parent strain. Under a same culture condition, a tryptophan production of the engineered strain is higher than a tryptophan production of the parent strain.

[0054] In some embodiments, the modification includes generating the pepD mutant protein by introducing a mutation at an amino acid site of the pepD protein of the parent strain, wherein the mutation includes at least one of mutations S21T, G225A, and A484K; or overexpressing a gene encoding the pepD protein or a gene encoding the pepD mutant protein using a high-copy plasmid as a vector; or in a genome of the parent strain, replacing the promoter of a gene encoding the pepD protein or the promoter of a gene encoding the pepD mutant protein with a strong promoter; or improving the stability of mRNA transcribed from a gene encoding the pepD protein or a gene encoding the pepD mutant protein.

[0055] In some embodiments, the pepD mutant protein includes a sequence having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 1.

[0056] In some embodiments, the modification includes: knocking out a gene encoding the fadR protein in the parent strain; or introducing a mutation at an amino acid site of the fadR protein of the parent strain, wherein the mutation includes at least one of mutations A140T and L171I; or in a genome of the parent strain, replacing the promoter of a gene encoding the fadR protein or the promoter of a gene encoding the fadR mutant protein with a weak promoter; or inhibiting translation efficiency or reducing stability of mRNA transcribed from a gene encoding the fadR protein or a gene encoding the fadR mutant protein. In some embodiments, the fadR mutant protein includes a sequence having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 2.

[0057] In some embodiments, the modification includes following operations.

[0058] The endogenous pepD and/or fadR proteins of the starting strain are directly modified, or the exogenous pepD and/or fadR proteins are modified before being introduced into the starting strain. The modification method is selected from at least one of the following techniques. [0059] a. In the parent strain, the modification of the pepD protein may be any one of the following. [0060] (a1) Introducing mutations at three amino acid sites of the pepD protein, including mutations S21T, G225A, and A484K, to obtain the pepD mutant protein, referred to as pepD.sup.S21T, G225A, A484K, and an amino acid sequence of the pepD mutant protein being shown in SEQ ID NO: 1. [0061] (a2) Overexpressing a gene encoding the pepD protein or a gene encoding the mutant protein pepD.sup.S21T, G225A, A484K using a high-copy plasmid as the vector. [0062] (a3) In a genome of the parent strain, replacing the promoter of a gene encoding the pepD protein or the promoter of a gene encoding the mutant protein pepD.sup.S21T, G225A, A484K with a strong promoter. [0063] (a4) Improving the stability of mRNA transcribed from a gene encoding the pepD protein or a gene encoding the mutant protein pepD.sup.S21T, G225A, A484K. [0064] (a5) Any manner or combination of manners capable of enhancing an expression level of a gene encoding the pepD protein or a gene encoding the mutant protein pepD.sup.S21T, G225A, A484K. [0065] b. In the parent strain, the modification of the fadR protein may be any one of the following. [0066] (b1) Knocking out a gene encoding the fadR protein in the parent strain. [0067] (b2) Introducing mutations at two amino acid sites of the gene encoding the fadR protein, including mutations A140T and L171I, to obtain the fadR mutant protein, referred to as fadR.sup.A140T, L171I, and an amino acid sequence of the fadR mutant protein being shown in SEQ ID NO: 2. [0068] (b3) In a genome of the parent strain, replacing the promoter of a gene encoding the fadR protein or the promoter of a gene encoding the mutant protein fadR.sup.A140T, L171I with a weak promoter. [0069] (b4) Inhibiting the translation efficiency or reducing the stability of mRNA transcribed from a gene encoding the fadR protein or a gene encoding the mutant protein fadR.sup.A140T, L171I. [0070] (b5) Any manner or combination of manners capable of weakening an expression level of a gene encoding the fadR protein or a gene encoding the mutant protein fadR.sup.A140T, L171I.

[0071] Further, in specific embodiments of the present disclosure, the parent strain is a tryptophan-producing strain; and the parent strain is selected from any one of Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, yeast cells, or the like. When the parent strain is Escherichia coli, it is further preferably selected from one of a strain IBEWQ, a mutant strain IBEWQ-62, a mutant strain IBEWQ-624, an Escherichia coli Nissle 1917, Escherichia coli BL21, Escherichia coli HB101, Escherichia coli JM109, Escherichia coli DH10B, or Escherichia coli MG1655.

[0072] In some embodiments, the engineered strain is the mutant strain IBEWQ-624. The mutant strain IBEWQ-624 includes a pepD mutant and a fadR mutant protein, the pepD mutant protein has an amino acid sequence shown in SEQ ID NO: 1, and the fadR mutant protein has an amino acid sequence shown in SEQ ID NO: 2.

[0073] In some embodiments, the obtaining the engineered strain includes culturing the parent strain in culture media including different concentrations of tryptophan and measuring a biomass in each culture medium; determining a growth rate of the parent strain based on the biomass; and obtaining the engineered strain capable of increasing a tryptophan production by fermenting a strain with a faster growth rate in a culture medium including high-concentration tryptophan.

[0074] In some embodiments, a concentration of the high-concentration tryptophan is in a range of 50 g/L to 70 g/L.

[0075] In specific embodiments of the present disclosure, the engineered strain IBEWQ-624 is constructed by knocking out a gene encoding the fadR protein in a genome of the strain IBEWQ, mutating a gene encoding the pepD protein into a gene encoding pepD.sup.S21T, G225A, A484K protein, and replacing the promoter of the gene encoding pepD.sup.S21T, G225A, A484K protein with a strong promoter PJ23119.

[0076] One of embodiments of the present disclosure provides a mutant protein. The mutant protein includes a sequence having at least 80% sequence identity to an amino acid sequence shown in SEQ ID NO: 1 or a sequence having at least 80% sequence identity to an amino acid sequence shown in SEQ ID NO: 2.

[0077] In some embodiments, the mutant protein includes a pepD mutant protein, and the pepD mutant protein includes at least one of mutations S21T, G225A, and A484K.

[0078] In some embodiments, the mutant protein includes a fadR mutant protein, and the fadR mutant protein includes at least one of mutations A140T and L171I.

[0079] One of embodiments of the present disclosure provides a DNA molecule. In some embodiments, the DNA molecule comprises a gene encoding the mutant protein described above.

[0080] One of embodiments of the present disclosure provides a gene expression cassette. In some embodiments, the gene expression cassette comprises the mutant protein described above or the gene encoding the mutant protein described above.

[0081] One of embodiments of the present disclosure provides a recombinant vector. In some embodiments, the recombinant vector comprises the mutant protein described above or the gene encoding the mutant protein described above.

[0082] One of embodiments of the present disclosure provides a use of an engineered strain in increasing tryptophan production.

[0083] In some embodiments, the use includes fermenting and culturing the engineered strain to obtain tryptophan with increased production.

[0084] In some embodiments, the pepD mutant protein included in the engineered strain includes any one of the following: (A1) a protein having an amino acid sequence shown in SEQ ID NO: 1; or (A2) a protein derived from the amino acid sequence shown in SEQ ID NO: 1 containing one or more amino acid substitutions, deletions, and/or insertions, and possessing the same function as the protein having an amino acid sequence shown in SEQ ID NO: 1; or (A3) a protein having an amino acid sequence with at least 99%, 95%, 90%, 85%, or 80% homology to any one of the amino acid sequences defined in (A1) and (A2), and possessing the same function as the proteins defined in (A1) and (A2); or (A4) a fusion protein obtained by linking a tag to the N-terminus and/or C-terminus of any one of the proteins defined in (A1) to (A3).

[0085] In some embodiments, the fadR mutant protein included in the engineered strain includes any one of the following: (A1) a protein having an amino acid sequence shown in SEQ ID NO: 2; or (A2) a protein derived from the amino acid sequence shown in SEQ ID NO: 2 containing one or more amino acid substitutions, deletions, and/or insertions, and possessing the same function as the protein having an amino acid sequence shown in SEQ ID NO: 2; or (A3) a protein having an amino acid sequence with at least 99%, 95%, 90%, 85%, or 80% homology to any one of the amino acid sequences defined in (A1) and (A2), and possessing the same function as the proteins defined in (A1) and (A2); or (A4) a fusion protein obtained by linking a tag to the N-terminus and/or C-terminus of any one of the proteins defined in (A1) to (A3).

[0086] In some embodiments, the gene encoding the pepD mutant protein includes a DNA molecule having at least 99%, 95%, 90%, 85%, or 80% homology to a DNA sequence defined by a gene encoding the pepD.sup.S21T, G225A, A484K mutant protein.

[0087] In some embodiments, the gene encoding the fadR mutant protein includes a DNA molecule having at least 99%, 95%, 90%, 85%, or 80% homology to a DNA sequence defined by a gene encoding the fadR.sup.A140T, L171I mutant protein.

[0088] The present disclosure provides any one of the following biomaterials, which may be used to increase tryptophan production. [0089] (I) Protein: pepD.sup.S21T, G225A, A484K mutant protein and/or fadR.sup.A140T, L171I mutant protein. [0090] (II) Gene: a gene encoding pepD.sup.S21T, G225A, A484K mutant protein and/or a gene encoding fadR.sup.A140T, L171I mutant protein. [0091] (III) Expression cassette: an expression cassette including a gene encoding pepD.sup.S21T, G225A, A484K mutant protein and/or a gene encoding fadR.sup.A140T, L171I mutant protein, or an expression cassette including a DNA fragment of the gene encoding the pepD.sup.S21T, G225A, A484K mutant protein and/or a DNA fragment of the gene encoding the fadR.sup.A140T, L171I mutant protein. [0092] (IV) Recombinant vector: a recombinant vector including a gene encoding pepD.sup.S21T, G225A, A484K mutant protein and/or a gene encoding fadR.sup.A140T, L171I mutant protein, or a recombinant vector including a DNA fragment of the gene encoding the pepD.sup.S21T, G225A, A484K mutant protein and/or a DNA fragment of the gene encoding the fadR.sup.A140T, L171I mutant protein. [0093] (V) Recombinant strain: a recombinant strain including a gene encoding pepD.sup.S21T, G225A, A484K mutant protein and/or a gene encoding fadR.sup.A140T, L171I mutant protein, or a recombinant strain including a DNA fragment of the gene encoding the pepD.sup.S21T, G225A, A484K mutant protein and/or a DNA fragment of the gene encoding the fadR.sup.A140T, L171I mutant protein. [0094] (a) A use of a biomaterial in increasing tryptophan production of a parent strain. [0095] (b) A use of a biomaterial in producing tryptophan. [0096] (c) A use of pepD.sup.S21T, G225A, A484K mutant protein or fadR.sup.A140T, L171I mutant protein in increasing tryptophan production of a parent strain. [0097] (d) A use of pepD.sup.S21T, G225A, A484K mutant protein or fadR.sup.A140T, L171I mutant protein in producing tryptophan.

EMBODIMENTS

[0098] The following provides a detailed description of specific embodiments of the present disclosure. However, it should be understood that the scope of protection of the present disclosure is not limited to these specific embodiments.

[0099] In the present disclosure, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents are commercially available.

Embodiment 1 Screening for a Tolerant Strain with Accelerated Growth Rate in Tryptophan Fermentation Broth

[0100] Fusion of cytidine deaminase with the a subunit of Escherichia coli RNA polymerase can accelerate mutations to levels that support efficient adaptive evolution in Escherichia coli without reducing cell viability.

[0101] Primers RNAP -F/R and CDA-F/R were used to amplify a gene sequence encoding the a subunit of Escherichia coli RNA polymerase and a gene sequence encoding the cytidine deaminase of a genome of the parent strain, respectively. Then, two end sequences were fused and expressed using the primers RNAP -F and CDA-R, digested with restriction enzymes EcoRI and NcoI, and then ligated into the temperature-sensitive plasmid pKD46 (GenBank accession no.: MF287367) that has been digested with the same enzymes, to obtain the plasmid denoted as pKAP, and the plasmid pKAP included an arabinose-inducible promoter regulating the expression of the gene of the a subunit of Escherichia coli RNA polymerase and the gene of the cytidine deaminase.

[0102] The primer sequences used for constructing the plasmid pKAP was as follows:

TABLE-US-00001 RNAP-F: SEQIDNO:7 GAATTCatgcagggttctgtgacag;. RNAP-R: SEQIDNO:8 CGATCCGCCACCGCCAGAGCCACCTCCGCCctcgtcagcgatgcttgcc ggtg;. CDA-F: SEQIDNO:9 GGCGGAGGTGGCTCTGGCGGTGGCGGATCGcatccacgttttcaaaccg c;. CDA-R: SEQIDNO:10 CCATGGttaagcgagaagcactcgg;.

Embodiment 2

(1) Construction of a Parent Strain IBEWQ

[0103] The parent strain IBEWQ was derived from Escherichia coli W3110 (competent cells of which were commercially available from various biological reagent suppliers). Specifically, at the tnaA locus, the trpE.sup.S40F, M1293T DCBA gene was expressed in tandem under the control of the tac promoter, at the trpR locus, AroF.sup.{circumflex over ()}P148L, Q152I, N8K, Aro.sup.GL76V, P150L, D146N, tktA, and ppsA genes derived from Escherichia coli K-12 were expressed, and at the tyrR locus, the SerA gene derived from Bacillus subtilis was expressed. At the same time, the promoters of the tyrA and pheA genes were replaced with the promoter PJ23114.

(2) Acquisition of a Mutant Strain IBEWQ-62

[0104] The plasmid pKAP was electrotransformed into the strain IBEWQ to generate a strain IBEWQ-pKAP. The strain IBEWQ-pKAP was used as the parent strain and continuously subculture and evolution were performed in seed media (supplemented with ampicillin resistance) including different concentrations of tryptophan. After 12 hours of incubation, each seed medium was diluted twofold, and the biomass in each seed medium was measured individually.

TABLE-US-00002 TABLE 1 Biomass measurement values under different concentrations of tryptophan Concentration of tryptophan Biomass OD.sub.600 (0 Biomass OD.sub.600 (15 in culture medium (g/L) mM L-arabinose) mM L-arabinose) 30 0.69 0.67 40 0.56 0.55 50 0.48 0.48 60 0.27 0.36 70 0.10 0.30

[0105] A strain exhibiting accelerated growth rate in a culture medium including high-concentration tryptophan was cultured in a fermentation medium at 37 C. to eliminate the plasmid pKAP. After plating and colony purification, a mutant strain with significantly improved tryptophan production was designated as IBEWQ-62.

[0106] Composition of a seed medium: 2.4 g/L K.sub.2HPO.sub.4, 9.6 g/L KH.sub.2PO.sub.4, 15 g/L yeast extract, 10 g/L rice bran, 5.0 g/L (NH.sub.4).sub.2SO.sub.4, 1.0 g/L MgSO.sub.4.Math.7H.sub.2O, and 20 g/L glucose, natural pH.

[0107] Composition of a shake flask fermentation medium: 20 g/L glucose, 3.0 g/L yeast extract powder, 30 g/L rice bran, 1.6 g/L (NH.sub.4).sub.2SO.sub.4, 2.0 g/L citric acid, 5.6 g/L K.sub.2HPO.sub.4, 2.0 g/L MgSO.sub.4.Math.7H.sub.2O, 80 mg/L FeSO.sub.4.Math.7H.sub.2O, 4.0 mg/L CoCl.sub.2.Math.6H.sub.2O, 0.6 mg/L CuSO.sub.4.Math.5H.sub.2O, 6.5 mg/L ZnSO.sub.4.Math.7H.sub.2O, 20 mg/L Na.sub.2SO.sub.4, 4.5 mg/L MnSO.sub.4.Math.H.sub.2O, and 20 g/L calcium carbonate, pH=7.2.

Embodiment 3 Tryptophan Produced by Fermentation of Mutant Strains and its Genomic Sequencing Analysis

[0108] The parent strain IBEWQ and the mutant strain IBEWQ-62 were subjected to fermentation. First, each strain preserved in glycerol was revived on slant agar, and then transferred into the seed medium to culture IBEWQ and IBEWQ-62 overnight. The obtained seeds of IBEWQ and IBEWQ-62 were inoculated into 500 mL shake flasks containing 100 mL of fermentation medium, followed by cultivation at 220 rpm and 37 C. Samples were collected to measure residual glucose content and tryptophan production. After 45 hours of fermentation, the tryptophan production level of IBEWQ-62 was significantly higher than that of the parent strain.

[0109] The genome of the mutant strain IBEWQ-62 was extracted and subjected to whole-genome sequencing. A comparison of genome analysis revealed that point mutations occurred in the IBEWQ-62 genome compared to the parent strain, and the mutations leading to amino acid mutations were summarized in Table 2.

TABLE-US-00003 TABLE2 MutationsinthegenomeofIBEWQ-62comparedtothewild-typestrain Gene name Mutation Sequenceaftermutation Sequencebeforemutation Mutated S21T, MSELSQLSPQPLWDIFAKICT MSELSQLSPQPLWDIFAKICsIPH pepD G225A, IPHPSYHEEQLAEYIVGWAK PSYHEEQLAEYIVGWAKEKGFH gene and EKGFHVERDQVGNILIRKPA VERDQVGNILIRKPATAGMENR A484K TAGMENRKPVVLQAHLDM KPVVLQAHLDMVPQKNNDTVH VPQKNNDTVHDFTKDPIQPY DFTKDPIQPYIDGEWVKARGTTL IDGEWVKARGTTLGADNGI GADNGIGMASALAVLADENVV GMASALAVLADENVVHGPL HGPLEVLLTMTEEAGMDGAFGL EVLLTMTEEAGMDGAFGLQ QGNWLQADILINTDSEEEGEIYM GNWLQADILINTDSEEEGEIY GCAGGIDFTSNLHLDREAVPAGF MGCAGGIDFTSNLHLDREA ETFKLTLKGLKGGHSGGEIHVGL VPAGFETFKLTLKGLKGGHS gNANKLLVRFLAGHAEELDLRLI GGEIHVGLaNANKLLVRFLA DFNGGTLRNAIPREAFATIAVAA GHAEELDLRLIDFNGGTLRN DKVDVLKSLVNTYQEILKNELA AIPREAFATIAVAADKVDVL EKEKNLALLLDSVANDKAALIA KSLVNTYQEILKNELAEKEK KSRDTFIRLLNATPNGVIRNSDV NLALLLDSVANDKAALIAKS AKGVVETSLNVGVVTMTDNNV RDTFIRLLNATPNGVIRNSD EIHCLIRSLIDSGKDYVVSMLDSL VAKGVVETSLNVGVVTMTD GKLAGAKTEAKGAYPGWQPDA NNVEIHCLIRSLIDSGKDYVV NSPVMHLVRETYQRLENKTPNI SMLDSLGKLAGAKTEAKGA QIIHAGLECGLFKKPYPEMDMVS YPGWQPDANSPVMHLVRET IGPTITGPHSPDEQVHIESVGHY YQRLENKTPNIQIIHAGLECG WILLTELLKEIPaK(SEQIDNO: LFKKPYPEMDMVSIGPTITG 4) PHSPDEQVHIESVGHYWTLL TELLKEIPKK(SEQIDNO:1) Mutated A140T MVIKAQSPAGFAEEYIIESIW MVIKAQSPAGFAEEYIIESIW fadRgene and NNRFPPGTILPAERELSELIGa NNRFPPGTILPAERELSELIGaTRT L171I TRTTLREVLQRLARDGWLTI TLREVLQRLARDGWLTIQHGKP QHGKPTKVNNFWETSGLNIL TKVNNFWETSGLNILETLARLD ETLARLDHESVPQLIDNLLS HESVPQLIDNLLSVRTNISTIFIRT VRTNISTIFIRTAFRQHPDKA AFRQHPDKAQEVLATANEVAD QEVLATANEVADHADIFAEL HADaFAELDYNIFRGLAFASGNP DYNIFRGLAFASGNPIYGLIL IYGLILNGMKGIYTRIGRHYFAN NGMKGIYTRIGRHYFANPEA PEARSLALGFYHKLSALCSEGAH RSLALGFYHKLSALCSEGAH DQVYETVRRYGHESGEIWHRM DQVYETVRRYGHESGEIWH QKNLPGDLAIQGR(SEQIDNO: RMQKNLPGDLAIQGR(SEQ 5) IDNO:2) Mutated V94Land MAISIKTPEDIEKMRVAGRL MAISIKTPEDIEKMRVAGRLAAE mapgene Q182N AAEVLEMIEPYVKPGVSTGE VLEMIEPYVKPGVSTGELDRICN LDRICNDYIVNEQHAVSACL DYIVNEQHAVSACLGYHGYPKS GYHGYPKSVCISINEVVCHG VCISINEVVCHGIPDDAKLLKDG IPDDAKLLKDGDIINIDVTVI DIVNIDVTVIKDGFHGDTSKMFI KDGFHGDTSKMFIVGKPTIM VGKPTIMGERLCRITQESLYLAL GERLCRITQESLYLALRMVK RMVKPGINLREIGAAIQKFVEAE PGINLREIGAAIQKFVEAEGF GFSVVREYCGHGIGRGFHEEPQ SVVREYCGHGIGRGFHEEPn VLHYDSRETNVVLKPGMTFTIEP VLHYDSRETNVVLKPGMTF MVNAGKKEIRTMKDGWTVKTK TIEPMVNAGKKEIRTMKDG DRSLSAQYEHTIVVTDNGCEILT WTVKTKDRSLSAQYEHTIV LRKDDTIPAIISHDE(SEQID VTDNGCEILTLRKDDTIPAIIS NO:6) HDE(SEQIDNO:3)

[0110] The mutation sites of the genes pepD, fadR, and map are as follows.

[0111] The mutated pepD gene, denoted as pepD.sup.S21T,G225A,A484K, includes three mutations S21T, G225A, and A484K.

[0112] The mutated fadR gene, denoted as fadR.sup.A140T,L171I, includes two mutations A140T and L171I.

[0113] The mutated map gene, denoted as map.sup.V94,Q182N, includes two mutations V94L and Q182N.

Embodiment 4 Impact of Mutated Genes on Fermentation Performance

[0114] In an original strain, the promoters of the amino acid sequence mutated genes (fadR, pepD, and map genes) were individually replaced with promoters of different strengths, and the impact of each mutant on tryptophan production in fermentation was compared.

[0115] Following standard gene editing procedures, the promoters of the genes pepD, fadR, and map in the strain IBEWQ were individually replaced with a strong promoter PJ23119. Similarly, following standard gene editing procedures, the promoters of the genes pepD, fadR, and map were replaced with a weak promoter PJ23114. The correctly verified strains after construction were activated in the seed medium for 12 to 16 hours and were transferred into the fermentation medium the next day.

[0116] After the fermentation was completed, the tryptophan production of each production strain was shown in Table 3.

TABLE-US-00004 TABLE 3 Modified protein IBEWQ IBEWQ-62 PJ23119 PJ23114 fadR 3.87 4.53 3.43 4.18 0.11 g/L 0.30 g/L 0.68 g/L 0.15 g/L pepD 4.06 3.68 0.69 g/L 0.80 g/L map 3.93 3.71 0.33 g/L 0.06 g/L

[0117] Based on the fermentation results, weakening of the fadR mutant gene significantly contributes to an increase in tryptophan production, whereas strengthening the pepD mutant gene is beneficial to increase tryptophan yield, and the map mutant has no noticeable impact on tryptophan production.

Embodiment 5: Overexpression of Mutated Genes or Construction of Weakened Plasmids

[0118] In the mutant strain IBEWQ-62, the promoter of the fadR.sup.A140T,L171I gene was replaced by PJ23114, denoted as a strain IBEWQ-621.

[0119] The promoter of the pepD.sup.S21T, G225A, A484K gene was replaced by PJ23119, denoted as a strain IBEWQ-622.

[0120] The fadR.sup.A140T,L171I gene was knocked out in a genome of the strain IBEWQ-622, denoted as a strain IBEWQ-623.

[0121] The tryptophan production results are shown in Table 1. It is evident that the enhanced expression of the pepD gene significantly improves the fermentation performance, while weakening or even completely knocking out the fadR gene yields the best improvement in fermentation performance. Based on this, a combined strain IBEWQ-624 was constructed by knocking out the fadR gene in the genome of the strain IBEWQ-62 and replacing the promoter of the pepD.sup.S21T,G225A,A484K gene by PJ23119.

[0122] The fermentation level of the strain IBEWQ-624 reaches 4.910.28 g/L, with a glucose-to-tryptophan yield of 24.5%. These results demonstrate that weakening the fadR gene is beneficial for improving the fermentation performance, with a complete knockout of the fadR gene representing the most effective strategy.

Embodiment 6 Scale-Up Validation

[0123] The parent strain IBEWQ and the mutant strain IBEWQ-624 were inoculated into 500 mL shake flasks containing 100 mL of seed medium, respectively, and cultured at 37 C. and 200 rpm for 12 to 16 hours, with an OD.sub.600 values of 11-13. The cultured seed solution was inoculated into a 5 L fermenter at a 10% (v/v) inoculation volume, with an initial aeration rate of 1.5 vvm and an initial agitation speed of 400 rpm. During fermentation, 25% ammonia water was fed into the fermenter to maintain the pH at 7.0. The fermentation temperature was controlled at 370.5 C., and the agitation speed and aeration rate were manually adjusted to maintain the dissolved oxygen level in a range of 20% to 30%. After inoculation for approximately 6 hours, a sharp increase in dissolved oxygen level was observed, indicating depletion of the initial glucose. The automatic feeding mode was then activated, and 800 g/L of glucose was fed to maintain the glucose concentration in the fermentation broth below 1 g/L.

[0124] After 16 hours of fermentation, samples were taken every 2 to 4 hours for analysis. At 48 hours, the tryptophan production of the parent strain IBEWQ reached 42.124.61 g/L, a glucose-to-tryptophan yield reached 19.1%, and the tryptophan production of the mutant strain IBEWQ-624 reached 62.385.80 g/L and the glucose-to-tryptophan yield reached 24.1%.

Embodiment 7 Use of Modification Manners in Other Strains

[0125] Taking Escherichia coli Nissle 1917 as a parent strain, in the genome of the parent strain, a fadR gene was knocked out, a pepD gene was mutated to pepD.sup.S21T,G225A,A484K, and the promoter of the pepD gene was replaced by PJ23119 to obtain a recombinant strain N-RD. After 40 hours of shake flask fermentation, 0.970.06 g/L of tryptophan was measured in the fermentation broth of the parent strain Escherichia coli Nissle 1917, while 1.280.03 g/L of tryptophan was measured in the fermentation broth of the recombinant strain, representing a 1.32-fold increase in the tryptophan production.

[0126] Composition of the shake flask fermentation medium: 10 g/L glucose, 5.0 g/L yeast extract powder, 10 g/L rice bran, 6.0 g/L (NH.sub.4).sub.2SO.sub.4, 3.0 g/L sodium citrate, 2.0 g/L L-glutamine, 1.0 g/L L-serine, 5.6 g/L K.sub.2HPO.sub.4, 3.0 g/L MgSO.sub.4.Math.7H.sub.2O, 65 mg/L FeSO.sub.4.Math.7H.sub.2O, and 20 g/L calcium carbonate, and pH=7.2.

[0127] Taking Escherichia coli BL21, HB101, JM109, DH10B, and MG1655 as the parent strain, a similar effect of 1.3 to 1.5-fold increase in tryptophan production can also be achieved.

[0128] The foregoing description only illustrates preferred embodiments of the present disclosure, but the scope of the present disclosure is not limited thereto. Those skilled in the art may readily conceive of various modifications or substitutions within the technical scope disclosed herein, all of which should be encompassed within the protection scope of the present disclosure. Therefore, the protection scope of the present invention shall be defined by the claims.