RhtA gene promoter and mutant for improving homoserine tolerance and their application

Abstract

This invention discloses an rhtA gene promoter, mutants, and their applications for enhancing homoserine tolerance, belonging to the field of bioengineering. Using error-prone PCR, an RhtA promoter and RhtA mutants with enhanced homoserine efflux capacity were screened and obtained. These mutants can confer enhanced homoserine stress tolerance to host bacteria. Compared to the wild-type RhtA in *E. coli*, the mutants exhibit one or more mutations at promoter position -268, such as thymine to cytosine, V43A, or A271V. The mutants can increase L-homoserine fermentation yield in *Bacillus coli* producing substrates by approximately 58%-77%. Therefore, the beneficial mutants provided by this invention can lay a solid foundation for the industrial-scale, efficient production of L-homoserine and its downstream metabolites.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, specifically relating to transport protein mutants that enhance high serine tolerance and their applications. Background Technology

[0002] L-homoserine, also known as homoserine or (2S)-2-amino-4-hydroxybutyric acid, appears as a white crystalline solid or powder. As a non-essential amino acid, L-homoserine is a common precursor to threonine and methionine. Although homoserine does not participate in protein synthesis, it is an important functional amino acid with significant roles in many fields, including biology, chemical engineering, and medicine.

[0003] Currently, the reported methods for producing L-homoserine can be broadly categorized into chemical synthesis, electrochemical reduction, and microbial fermentation. Microbial fermentation has long been widely used in the amino acid production industry.

[0004] In the design and construction of efficient chassis cell factories, the identification and engineering of transport proteins are effective ways to improve host cell growth and the potency of target products. Identifying and screening enhanced efflux transport proteins to reduce the intracellular concentration of products is beneficial for minimizing intracellular metabolic inhibition and improving biomanufacturing efficiency. Studies have found that enhancing the expression of L-threonine transporter genes, especially the RhtA-encoding gene rhtA, can significantly increase the biomass of *E. coli* under L-homoserine stress, suggesting that RhtA may be the main L-homoserine efflux protein in *E. coli*. Summary of the Invention

[0005] The purpose of this invention is to provide a mutant of L-homoserine RhtA, its encoding gene, recombinant vector, and its application in the production of L-homoserine.

[0006] The amino acid sequence of homoserine RhtA before mutation is shown in SEQ ID No. 1:

[0007] MPGSLRKMPVWLPIVILLVAMASIQGGASLAKSLFPLVGAPGVTALRLALGTLILIAFFKPWRLRFAKEQRLPLLFYGVSLGGMNYLFYLSIQTVPLGIAVALEFTGPLAVALFSSRRPVDFVWVVLAVLGLWFLLPLGQDVSHVDLT GCALALGAGACWAIYILSGQRAGAEHGPATVAIGSLIAALIFVPIGALQAGEALWHWSVIPLGLAVAILSTALPYSLEMIALTRLPTRTFGTLMSMEPALAAVSGMIFLGETLTPIQLLALGAIIAASMGSTLTVRKESKIKELDIN*

[0008] The gene sequence encoding homoserine RhtA before mutation is shown in SEQ ID No. 2:

[0009] atgcctggttcattacgtaaaatgccggtctggttaccaatagtcatattgctcgttgccatggcgtctattcagggtggagcctcgttagctaagtcactttttcctctggtgggcgcaccgggtgtcactgcgctgcgtctggcattaggaacgctgatcctcatcgcgttctttaagccatggcgactgcgctttgccaaagagcaacggttaccgctgttgttttacggcgtttcgctgggtgggatgaattatcttttttatctttctattcagacagtaccgctgggtattgcggtggcgctggagttcaccggaccactggcggtggcgctgttctcttctcgtcgcccggtagatttcgtctgggttgtgctggcggttcttggtctgtggttcctgctaccgctggggcaagacgtttcccatgtcgatttaaccggctgtgcgctggcactgggggccggggcttgttgggctatttacattttaagtgggcaacgcgcaggagcggaacatggccctgcgacggtggcaattggttcgttgattgcagcgttaattttcgtgccaattggagcgcttcaggctggtgaagcactctggcactggtcggttattccattgggtctggctgtcgctattctctcgaccgctctgccttattcgctggaaatgattgccctcacccgtttgccaacacggacatttggtacgctgatgagcatggaaccggcgctggctgccgtttccgggatgattttcctcggagaaacactgacacccatacagctactggcgctcggcgctatcatcgccgcttcaatggggtctacgctgacagtacgcaaagagagcaaaataaaagaattagacattaattaa。

[0010] There is a promoter sequence upstream of SEQ ID No.2. The promoter sequence before mutation is shown in SEQ ID No.3:

[0011] ttgtgatgtct aacgggccatttcatgtaacagaacgtttccatacaccgctatccatctaaatttaaatcactttttcagagaactgcgtaagtattacgcatgttttccctgtcattcatccagattattcctaatcaccaga ctaatgattccatcaatcctggcgcattttagtcaaaacgggggaaaattttttcaacaaatgctcaaccagcattgggtatatccagtacactccacgctttacttaagtctagatatttgtgggagaaagg.

[0012] The inventors discovered that the promoter nucleotide sequence formed by mutating thymine to cytosine at position -268 of the nucleotide sequence shown in SEQ ID NO:3 can enhance the host bacterium's tolerance to hyperserine stress. The nucleotide sequence of the promoter following the mutant is shown in SEQ ID NO:4.

[0013] ttgtgatgtc c aacgggccatttcatgtaacagaacgtttccatacaccgctatccatctaaatttaaatcactttttcagagaactgcgtaagtattacgcatgttttccctgtcattcatccagattattcctaatcaccag actaatgattccatcaatcctggcgcattttagtcaaaacgggggaaaattttttcaacaaatgctcaaccagcattgggtatatccagtacactccacgctttacttaagtctagatatttgtgggagaaagg

[0014] The inventors discovered that mutating certain amino acid mutation sites in the amino acid sequence shown in SEQ ID NO.1 can increase the stress tolerance of the host bacteria.

[0015] The amino acid mutation site includes at least one of mutation point a and mutation point b;

[0016] Mutation point a: The 43rd position of the amino acid sequence shown in SEQ ID NO.1 is mutated to valine (V) and alanine (A);

[0017] Mutation point b: The amino acid sequence shown in SEQ ID NO.1 is mutated at position 271 from alanine A to valine V.

[0018] The above mutations can be obtained through methods such as mutagenesis, PCR site-directed mutagenesis, and / or homologous recombination.

[0019] When introducing the mutated nucleotide sequence into a recombinant vector constructed from a plasmid, the plasmid can be a TRP plasmid. Specifically, the mutated nucleotide sequence and a linearized fragment of the plasmid can be ligated to construct a recombinant vector.

[0020] The beneficial effects achieved by this invention are as follows: This invention utilizes error-prone PCR to screen and obtain various RhtA mutants with enhanced homoserine stress resistance. Compared to the wild-type RhtA in *E. coli*, these mutants exhibit one or more mutations among T(-268)C, V43A, and A271V. In specific embodiments, overexpression of these mutants increases the fermentation yield of L-homoserine-producing chassis strains by approximately 58%-77%. Therefore, the beneficial mutants provided by this invention, compared to the unmutated wild-type strains, are advantageous for producing high concentrations of L-homoserine, and the strains exhibit good stability, further reducing production costs as L-homoserine-producing strains. Attached Figure Description

[0021] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0022] Figure 1 Growth tolerance test of RhtA single mutant;

[0023] Figure 2 : Fermentation analysis of homoserine in RhtA single mutant;

[0024] Figure 3 Growth tolerance test of RhtA combined mutant;

[0025] Figure 4 : Fermentation analysis of high-serine content in RhtA combinatorial mutants;

[0026] Figure 5 : RhtA mutant transport capacity test;

[0027] Figure 6 RhtA was applied to the fermentation analysis of Corynebacterium glutamicum. Detailed Implementation

[0028] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0029] Example 1: Construction of the rhtA gene mutant library

[0030] The rhtA gene encoding the gene rhtA and its promoter were randomly mutagenized using error-prone PCR to construct a rhtA gene mutant library.

[0031] The primer sequences used in this embodiment are as follows:

[0032] P1:CTTTGATATACGCCGAGATCACGCGGATCCTTAATGGGAAACTC

[0033] P2:GAGAATTGACTCTAGAGCACTGACGTCGACCTGCAGAATAAATATG

[0034] P3:TGCTCTAGAGTCAATTCTCATG

[0035] P4:TGATCTCGGCGTATATCAAAGC

[0036] In this example, the ClonExpress One-Step Cloning Kit (Vazyme Biotech, China) was used to construct the corresponding gene expression plasmid. Using rTaq DNA polymerase (Takara Bio Inc, China), and with the *E. coli* W3110 genome as a template, the randomly mutated rhtA gene and its promoter fragment were amplified via error-prone PCR using primers P1 and P2. Using high-fidelity DNA polymerase, and with the TRP plasmid as a template, the TRP plasmid backbone was obtained via PCR using primers P3 and P4. The mutated rhtA gene and its promoter fragment obtained by PCR were ligated to the TRP plasmid backbone using the ClonExpress One-Step Cloning Kit and then transformed into *E. coli* JM109 to obtain the rhtA mutant library. The resulting mutant plasmid was transformed into the homoserine-producing chassis strain *E. coli* HOM-1, and the L-homoserine yield was measured after 48 hours of fermentation to determine the translocation efficiency of the rhtA mutant. The E. coli HOM-1 mentioned above refers to E. coli W3110Δyjip::thrA (C1034T) BC-ppc-aspA-pntAB-asd, ΔycjX::thrA (C1034T) BC-ppc-aspA-pntAB-asd, ΔyeeL::thrA (C1034T) BC-aspC-asd、ΔyjgX::thrA (C1034T)BC-aspC-asd, pgi(ATG-GTG), Δtdh, ΔsstT, ΔiclR, ΔlysA, ΔmetA, ΔthrB.

[0037] Table 1

[0038]

[0039]

[0040] WT was E. coli HOM-1TRP-PrhtA, and the biomass was obtained by growing the engineered strain containing the mutant plasmid in M9-glucose medium in 96-well plates for 24 h. As shown in Table 1, the L-homoserine production of the RhtA mutant in experiments 5, 6, and 15 was significantly increased compared to WT. Therefore, these mutation sites were selected for further validation.

[0041] Example 2: Preparation and Identification of Beneficial Mutation Sites of Homoserine RhtA

[0042] In this example, recombinant plasmid TRP-PrhtA was constructed. (T(-268)C) TRP-PrhtA (T(-17)C) TRP-PrhtA (V43A) TRP-PrhtA (C(-120)T) TRP-PrhtA (P263S) TRP-PrhtA(A271V)

[0043] Using the wild-type *Escherichia coli* W3110 genome as a template, the rhtA gene and its promoter fragment were amplified by PCR using primers P1 and P2. Using the TRP plasmid as a template, the TRP plasmid backbone was obtained by PCR using primers P3 and P4. The mutated rhtA gene and its promoter fragment obtained by PCR were ligated to the TRP plasmid backbone using the ClonExpress one-step directional cloning seamless cloning kit to obtain the recombinant plasmid TRP-PrhtA.

[0044] Using recombinant plasmid TRP-PrhtA as a template, PCR was performed with primers P5 and P6 to induce a mutation at the -268 site, primers P7 and P8 to induce a mutation at the -17 site, primers P9 and P10 to induce a mutation at the V43 site, primers P11 and P12 to induce a mutation at the -120 site, primers P13 and P14 to induce a mutation at the P263 site, and primers P15 and P16 to induce a mutation at the A271 site. The point mutation PCR products were purified by agarose gel electrophoresis, digested with DpnI, and transformed into *E. coli* JM109. Transformants were selected and sent to a sequencing company for sequencing identification. Vectors containing the correct point mutations were reserved for later use. Simultaneously, each mutant plasmid was transformed into a homoserine-producing strain for later use.

[0045] The primer sequences used in this embodiment are as follows:

[0046] P5: ATTTGTGATGTCCAACGGGCCATT

[0047] P6: CGTTGGACATCACAAATCGCGAAGAG

[0048] P7: GTCTAGACATTGTGGGAGAAAGGATGCCT

[0049] P8: CCTTTTCCCACAAAATGTCTAGACTTAAG

[0050] P9: CGGGTGCCACTGCGCTGCGTC

[0051] P10: AGTGGCACCCGGTGCGCCCA

[0052] P11: GATTCATTAATCCTGGCGCATTTTAGTCA

[0053] P12:GCGCCAGGATTAATGGAATCATTAG

[0054] P13: CACTGACATCCATACAGCTACTGG

[0055] P14:GCTGTATGGATGTCAGTGTTTCTC

[0056] P15:GCTCGGCGTTATCATCGCCGCTTCAATG

[0057] P16:GATGATAACGCCGAGCGCCAGTAG

[0058] The engineered strains constructed above were inoculated into M9-glucose medium and TPM1 medium containing 1.0 g / L L-homoserine, respectively, and their growth capacity and fermentation performance were determined. The results are as follows: Figure 1 and Figure 2 As shown. The biomass of the recombinant strains containing the above mutants under a pressure of 1.0 g / L L-homoserine was 0.76, 0.54, 1.27, 0.52, 0.51, and 1.04, respectively. The T(-268)C, V43A, and A271V mutations showed significant improvements, increasing biomass by approximately 1.40 times, 2.35 times, and 1.92 times, respectively, compared to recombinant strains containing wild-type natural L-homoserine. The L-homoserine yield of the recombinant strains containing the above mutants was 8.73 g / L, 4.34 g / L, 8.21 g / L, 5.03 g / L, 5.31 g / L, and 8.35 g / L, respectively. The T(-268)C, V43A, and A271V mutations showed significant improvements, increasing biomass by approximately 68%, 58%, and 62%, respectively, compared to recombinant strains containing wild-type natural L-homoserine transporters.

[0059] Example 3: Combination mutations at beneficial mutation sites enhance RhtA activity and L-homoserine fermentation performance.

[0060] To further enhance RhtA activity, the beneficial mutation sites T(-268)C, V43A, and A271V from the above examples were combined with mutants. Three double-mutant combinations and one triple-mutant combination were constructed: T(-268)C / V43A, T(-268)C / A271V, V43A / A271V, and T(-268)C / V43A / A271V. The constructed combined mutant strains were inoculated into M9-glucose medium and TPM1 medium containing 1.0 g / L L-homoserine, respectively, and their growth capacity and fermentation performance were measured. The results are as follows: Figure 3 and Figure 4 As shown. The biomass of the recombinant strains containing the above mutants under a 1.0 g / L L-homoserine pressure was 0.74, 0.71, 1.38, and 0.71, respectively, which were approximately 1.35-fold, 1.31-fold, 2.50-fold, and 1.31-fold higher than those of the recombinant strains containing the wild-type natural L-homoserine transporter. The L-homoserine production of the recombinant strains containing the above mutants was 9.15 g / L, 8.84 g / L, 9.07 g / L, and 6.78 g / L, respectively. Among them, the three mutants T(-268)C / V43A, T(-268)C / A271V, and V43A / A271V showed significant improvements, increasing by approximately 77%, 71%, and 75%, respectively, compared to the recombinant strains containing the wild-type natural L-homoserine transporter.

[0061] The M9-glucose culture medium comprises: glucose 4 g / L, Na2HPO4·7H2O 12.8 g / L, KH2PO4 3 g / L, NaCl 0.5 g / L, NH4Cl 1 g / L, MgSO4 0.241 g / L, and CaCl2 0.011 g / L. The TPM1 culture medium comprises: glucose 20 g / L, yeast extract 4 g / L, MgSO4·7H2O 2 g / L, KH2PO4 4 g / L, (NH4)2SO4 14 g / L, L-lysine 0.164 g / L, L-threonine 0.238 g / L, L-methionine 0.149 g / L, trace element stock solution 5 mL, and CaCO3 10 g / L. Trace element mother liquor composition: FeSO4·7H2O 10g / L, CaCl2 1.35g / L, ZnSO4·7H2O 2.25g / L, MnSO4·4H2O 0.5g / L, CuSO4·5H2O 1g / L, (NH4)6Mo7O 24 ·4H2O 0.106g / L, Na2B4O7·10H2O 0.23g / L, 35% HCl, 10mL.

[0062] Example 4: Detection of transport capacity of various homoserine RhtA mutants

[0063] The recombinant plasmids containing mutants T(-268)C, V43A, A271V, T(-268)C / V43A, T(-268)C / A271V, V43A / A271V, and T(-268)C / V43A / A271V, constructed in the above examples, were transformed into homoserine-producing strains and cultured to the late logarithmic growth phase. Then, 20 g / L L-homoserine was added for 6 hours of treatment. The homoserine-treated cells were collected and washed three times with ice-cold M9 buffer. Cells were then suspended in preheated M9 medium supplemented with 40 μM thiamine hydrochloride to initiate efflux, and samples were taken for analysis. The rate of L-homoserine efflux (mg / g DCW) was indirectly represented by the rate of change in L-homoserine concentration per gram of dry cell weight (DCW). The results are shown below. Figure 5As shown, at 10 minutes, the homoserine content in suspensions containing the RhtA mutants T(-268)C, V43A, A271V, T(-268)C / V43A, T(-268)C / A271V, V43A / A271V, and T(-268)C / V43A / A271V changed significantly, reaching 13.17 mg / g DCW, 7.87 mg / g DCW, 9.45 mg / g DCW, 26.52 mg / g DCW, 18.76 mg / g DCW, 21.17 mg / g DCW, and 6.33 mg / g DCW, respectively. These values ​​were approximately 2.57 times, 1.54 times, 1.85 times, 5.18 times, 3.66 times, 4.14 times, and 1.24 times higher than those in recombinant bacteria containing wild-type natural L-homoserine RhtA.

[0064] Example 5: Application of the homoserine RhtA beneficial mutant to Corynebacterium glutamicum

[0065] In this example, the homoserine RhtA mutants T(-268)C, V43A, A271V, T(-268)C / V43A, T(-268)C / A271V, V43A / A271V, and T(-268)C / V43A / A271V constructed in the above examples were transformed into L-homoserine-producing Corynebacterium glutamicum 13032ΔthrB and Δddh::P, respectively. tac -lysC (C1055T) pXMJ19-thrA (C1034T) In this study, the fermentation performance of each engineered strain was determined by inoculating it into the fermentation medium. The results are as follows: Figure 6 As shown, the L-homoserine production of engineered strains containing mutants T(-268)C, V43A, A271V, T(-268)C / V43A, T(-268)C / A271V, V43A / A271V, and T(-268)C / V43A / A271V were 9.49 g / L, 9.09 g / L, 9.22 g / L, 10.26 g / L, 9.74 g / L, 10.14 g / L, and 8.17 g / L, respectively, which were approximately 45%, 39%, 40%, 57%, 49%, 55%, and 25% higher than those of recombinant strains containing wild-type natural L-homoserine transporters.

[0066] The fermentation medium components include: glucose 50 g / L, corn steep liquor 20 g / L, (NH4)2SO4 20 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.5 g / L, MnSO4·H2O 0.01 g / L, FeSO4·7H2O 0.01 g / L, vitamin B1 1 mg / L, vitamin B6 1 mg / L, biotin 0.025 mg / L, and vitamin B12 4 mg / L.

[0067] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A promoter for the RhtA gene, the nucleotide sequence of which is shown in SEQ ID NO:

4.

2. A mutant of RhtA, wherein the mutation site is the 271st position of the amino acid sequence shown in SEQ ID NO. 1, where alanine A is mutated to valine V.

3. An expression cassette comprising the gene encoding the promoter of claim 1 and / or the mutant of RhtA of claim 2.

4. An expression cassette comprising the promoter of claim 1 and the coding gene of a mutant of RhtA, wherein the mutant of RhtA is mutated at position 43 of the amino acid sequence shown in SEQ ID NO.1 by changing valine V to alanine A.

5. A recombinant vector comprising the coding gene of the promoter of claim 1 and / or the mutant of RhtA of claim 2.

6. The recombinant vector as described in claim 5, characterized in that, The plasmid was constructed by introducing the nucleotide sequence of the promoter shown in SEQ ID NO:4 and / or the nucleotide sequence of the mutant of RhtA as described in claim 2.

7. A recombinant strain comprising the recombinant vector of claim 6.

8. The recombinant strain according to claim 7, characterized in that, The plasmid vector is the TRP plasmid.

9. The use of the promoter according to claim 1 in increasing L-homoserine production or in the preparation of L-homoserine.

10. The use of the recombinant strain according to claim 7 or 8 in increasing L-homoserine yield or in the preparation of L-homoserine.

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