RhtA gene promoter for improving homoserine tolerance, mutant and application of rhtA gene promoter

Abstract

The invention discloses an rhtA gene promoter for improving homoserine tolerance, a mutant and application of the rhtA gene promoter and the mutant, and belongs to the field of bioengineering. An RhtA promoter with enhanced homoserine efflux ability and an RhtA mutant are obtained through screening by using an error-prone PCR method, host bacteria can be endowed with enhanced homoserine stress tolerance ability, and compared with escherichia coli wild type RhtA, the mutant has one or more mutations of a promoter 268 site basic group mutated from thymine to cytosine, V43A and A271V. The L-homoserine fermentation yield of the mutant produced chassis bacteria can be increased by about 58%-77%. Therefore, the beneficial mutant provided by the invention can lay a good foundation for industrial efficient production of L-homoserine and downstream metabolites.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering, and in particular relates to a transporter mutant capable of improving homoserine tolerance and an application thereof. Background Art

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

[0003] At present, the reported L-homoserine production methods can be roughly divided 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 transporters are effective ways to improve host cell growth and target product titer. Mining and screening for enhanced efflux transporters to reduce the intracellular concentration of products is beneficial to minimize intracellular metabolic inhibition and improve biomanufacturing efficiency. Studies have found that by enhancing the expression of L-threonine transporter genes, especially the RhtA encoding gene rhtA, the biomass of Escherichia coli under L-homoserine stress can be significantly increased, suggesting that RhtA may be the main L-homoserine efflux protein in Escherichia coli. Summary of the invention

[0005] The purpose of the present invention is to provide a mutant of L-homoserine RhtA, a coding gene, a recombinant vector and the application thereof 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 coding gene sequence of 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 found that the promoter nucleotide sequence formed by mutating the -268th base of the nucleotide sequence shown in SEQ ID NO:3 from thymine to cytosine can increase the ability to confer enhanced homoserine stress tolerance to the host bacteria. The nucleotide sequence of the promoter after the mutation is shown in SEQ ID NO:4.

[0013] ttgtgatgtc c aacgggccatttcatgtaacagaacgtttccatacaccgctatccatctaaatttaaatcactttttcagagaactgcgtaagtattacgcatgttttccctgtcattcatccagattattcctaatcaccag actaatgattccatcaatcctggcgcattttagtcaaaacgggggaaaattttttcaacaaatgctcaaccagcattgggtatatccagtacactccacgctttacttaagtctagatatttgtgggagaaagg

[0014] The inventors have found that mutating some of the 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 from valine V to alanine A;

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

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

[0019] When the mutated nucleotide sequence is introduced into a recombinant vector constructed by a plasmid, the plasmid may be a TRP plasmid. Specifically, the mutated nucleotide sequence and the linearized fragment of the plasmid may be connected to form a recombinant vector.

[0020] The beneficial effect achieved by the present invention is: the present invention uses the error-prone PCR method to screen and obtain a variety of RhtA mutants with enhanced homoserine stress ability, and relative to the wild-type rhtA of Escherichia coli, there are one or more mutations in T(-268)C, V43A, and A271V. In a specific embodiment, the above mutants are overexpressed respectively, and the fermentation yield of the chassis strain producing L-homoserine can be increased by about 58%-77%. Therefore, compared with the unmutated wild-type strain, the beneficial mutant provided by the present invention is conducive to the production of high-concentration L-homoserine, and the strain has good stability. As an L-homoserine production strain, it can further reduce production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0022] Figure 1 : Tolerant growth test of RhtA single mutant;

[0023] Figure 2 : Homoserine fermentation analysis of RhtA single mutants;

[0024] Figure 3 : Tolerance growth test of RhtA combination mutants;

[0025] Figure 4 : Homoserine fermentation analysis of RhtA combination mutants;

[0026] Figure 5 : Transport ability test of RhtA mutant;

[0027] Figure 6 :Application of RhtA in fermentation analysis of Corynebacterium glutamicum. DETAILED DESCRIPTION

[0028] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0029] Example 1 Construction of rhtA gene mutant library

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

[0031] The primer sequences used in this example are:

[0032] P1:CTTTGATATACGCCGAGATCACGCGGATCCTTAATGGGAAACTC

[0033] P2:GAGAATTGACTCTAGAGCACTGACGTCGACCTGCAGAATAAATATG

[0034] P3:TGCTCTAGAGTCAATTCTCATG

[0035] P4:TGATCTCGGCGTATATCAAAGC

[0036] In this example, the ClonExpress one-step directional cloning seamless cloning kit (ClonExpressIIOne Step Cloning kit, Vazyme Biotech, China) was used to construct the corresponding gene expression plasmid, and r TaqDNA polymerase (Takara Bio Inc, China) was used. The E. coli W3110 genome was used as a template, and primers P1 and P2 were used to amplify the randomly mutated rhtA gene and its promoter fragment by error-prone PCR. High-fidelity DNA polymerase was used, and the TRP plasmid was used as a template. The TRP plasmid skeleton was obtained by PCR using primers P3 and P4. The mutated rhtA gene and its promoter fragment obtained by PCR were connected to the TRP plasmid skeleton using the ClonExpress one-step directional cloning seamless cloning kit and then transferred to E. coli JM109 to obtain the rhtA mutant library. The resulting mutant plasmid was transferred to the homoserine production chassis strain E. coli HOM-1, and the L-homoserine production was measured after 48h fermentation to determine the transport effect of the rhtA mutant. The E. coli HOM-1 is E. coli W3110Δyjip::thrA (C1034T) BC-ppc-aspA-pntAB-asd, ΔycjX::thrA (C1034T) BC-ppc-aspA-pntAB-asd,ΔyeeL::thrA (C1034T) BC-aspC-asd, ΔyigX::thrA (C1034T)BC-aspC-asd, pgi(ATG-GTG), Δtdh, ΔsstT, ΔiclR, ΔlysA, ΔmetA, ΔthrB.

[0037] Table 1

[0038]

[0039] Among them, WT is E. coli HOM-1TRP-PrhtA, and the biomass is obtained by growing the engineered strain containing the mutant plasmid in a 96-deep well plate using M9-glucose medium for 24 hours. As shown in Table 1, the L-homoserine production of the rhtA mutants in Experiments 5, 6 and 15 was significantly improved compared with WT, so these mutation sites were selected for subsequent verification.

[0040] Example 2 Preparation and identification of beneficial mutation sites of homoserine RhtA

[0041] In this example, the 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) .

[0042] The wild-type E. coli W3110 genome was used as a template, and the rhtA gene and its promoter fragment were amplified by PCR using primers P1 and P2. The TRP plasmid skeleton was obtained by PCR using primers P3 and P4. The ClonExpress one-step directional cloning seamless cloning kit was used to connect the mutated rhtA gene and its promoter fragment obtained by PCR with the TRP plasmid skeleton to obtain the recombinant plasmid TRP-PrhtA.

[0043] Using the recombinant plasmid TRP-PrhtA as a template, the -268 site mutation was performed by PCR using primers P5 and P6, the -17 site mutation was performed by PCR using primers P7 and P8, the V43 site mutation was performed by PCR using primers P9 and P10, the -120 site mutation was performed by PCR using primers P11 and P12, the P263 site mutation was performed by PCR using primers P13 and P14, and the A271 site mutation was performed by PCR using primers P15 and P16. The point mutation PCR product was separated and purified by agarose gel and then digested with Dpn Ι and transferred to Escherichia coli E.coli JM109. The transformant was picked and sent to a sequencing company for sequencing identification, and the vector containing the correct point mutation was used for standby. At the same time, each mutant plasmid was transferred to a homoserine production strain for standby.

[0044] The primer sequences used in this example are:

[0045] P5:ATTTGTGATGTCCAACGGGCCATT

[0046] P6: CGTTGGACATCACAAATCGCGAAGAG

[0047] P7: GTCTAGACATTGTGGGAGAAAGGATGCCT

[0048] P8: CCTTTTCCCACAAAATGTCTAGACTTAAG

[0049] P9: CGGGTGCCACTGCGCTGCGTC

[0050] P10:AGTGGCACCCGGTGCGCCCA

[0051] P11: GATTCATTAATCCTGGCGCATTTTAGTCA

[0052] P12:GCGCCAGGATTAATGGAATCATTAG

[0053] P13: CACTGACATCCATACAGCTACTGG

[0054] P14:GCTGTATGGATGTCAGTGTTTCTC

[0055] P15:GCTCGGCGTTATCATCGCCGCTTCAATG

[0056] P16:GATGATAACGCCGAGCGCCAGTAG

[0057] The engineered strain constructed above was inoculated into M9-glucose medium and TPM1 medium containing 1.0 g / L L-homoserine to measure its growth ability and fermentation performance. The results are as follows: Figure 1 and Figure 2 As shown. The biomass of the recombinant strain containing the above mutants under 1.0g / L L-homoserine pressure was 0.76, 0.54, 1.27, 0.52, 0.51, and 1.04, respectively. Among them, the three mutation points T(-268)C, V43A, and A271V had obvious effects, which were about 1.40 times, 2.35 times, and 1.92 times higher than the recombinant bacteria containing wild-type natural L-homoserine, respectively. The L-homoserine production of the recombinant strain containing the above mutants was 8.73g / L, 4.34g / L, 8.21g / L, 5.03g / L, 5.31g / L, and 8.35g / L, respectively. Among them, the three mutation points T(-268)C, V43A, and A271V had obvious effects, which were about 68%, 58%, and 62% higher than the recombinant bacteria containing wild-type natural L-homoserine transporter, respectively.

[0058] Example 3 Combination mutation of beneficial mutation sites improves RhtA activity and L-homoserine fermentation performance

[0059] In order to further improve the activity of RhtA, the beneficial mutation sites T(-268)C, V43A, and A271V in the above example were combined and mutated. Three double mutation combinations and one triple mutation combination were constructed, namely T(-268)C / V43A, T(-268)C / A271V, V43A / A271V, and T(-268)C / V43A / A271V. The combined mutant engineering strains constructed above were inoculated into M9-glucose medium and TPM1 medium containing 1.0 g / L L-homoserine to measure their growth ability and fermentation performance. The results are shown in Figure 2. Figure 3 and Figure 4 As shown. The biomass of the recombinant strains containing the above mutants under 1.0g / L L-homoserine pressure were 0.74, 0.71, 1.38, and 0.71, respectively, which were about 1.35 times, 1.31 times, 2.50 times, and 1.31 times higher than those of the recombinant bacteria containing the wild-type natural L-homoserine transporter. The L-homoserine production of the recombinant strains containing the above mutants was 9.15g / L, 8.84g / L, 9.07g / L, and 6.78g / L, respectively, among which the three mutants T(-268)C / V43A, T(-268)C / A271V, and V43A / A271V had obvious improvements, which were about 77%, 71%, and 75% higher than those of the recombinant bacteria containing the wild-type natural L-homoserine transporter, respectively.

[0060] The components of the M9-glucose medium include: 4 g / L glucose, 12.8 g / L Na2HPO4·7H2O, 3 g / L KH2PO4, 0.5 g / L NaCl, 1 g / L NH4Cl, 0.241 g / L MgSO4, and 0.011 g / L CaCl2. The components of the TPM1 medium include: 20 g / L glucose, 4 g / L yeast powder, 2 g / L MgSO4·7H2O, 4 g / L KH2PO4, 14 g / L (NH4)2SO4, 0.164 g / L L-lysine, 0.238 g / L L-threonine, 0.149 g / L L-methionine, 5 mL trace element mother solution, and 10 g / L CaCO3. Trace element mother solution composition: FeSO4·7H2O 10g / L, CaCl2 1.35g / L, ZnSO4·7H2O 2.25g / L, MnSO4·4H2O0.5g / L, CuSO4·5H2O 1g / L, (NH4)6Mo7O 24 ·4H2O 0.106g / L, Na2B4O7·10H2O 0.23g / L, 35% HCl, 10mL.

[0061] Example 4 Detection of transport capacity of homoserine RhtA mutants

[0062] 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 respectively transferred into homoserine-producing strains, cultured to the end of logarithmic growth, and treated with 20 g / L of L-homoserine for 6 hours. The homoserine-treated bacteria were collected and washed three times with ice-cold M9 buffer. The cells were suspended in M9 medium preheated with 40 μM thiamine hydrochloride to start efflux, and sampling was performed for detection. The rate of change of L-homoserine concentration per gram of cell dry weight (DCW) indirectly represents the rate of L-homoserine efflux (mg / g DCW), and the results are shown as follows. Figure 5At 10 minutes, the homoserine content in the suspension 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, which were 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, which were increased by about 2.57 times, 1.54 times, 1.85 times, 5.18 times, 3.66 times, 4.14 times, and 1.24 times compared with the recombinant bacteria containing the wild-type natural L-homoserine RhtA.

[0063] Example 5 Application of the beneficial homoserine RhtA mutant to Corynebacterium glutamicum

[0064] 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 respectively transferred into L-homoserine-producing Corynebacterium glutamicum 13032ΔthrB, Δddh::P tac -lysC (C1055T) 、pXMJ19-thrA (C1034T) In the experiment, each engineered strain was inoculated into the fermentation medium to measure its fermentation performance. Figure 6 As shown, the L-homoserine production of the 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 the recombinant bacteria containing the wild-type natural L-homoserine transporter.

[0065] The fermentation medium components include: 50 g / L glucose, 20 g / L corn steep liquor, 20 g / L (NH4)2SO4, 1 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 0.01 g / L MnSO4·H2O, 0.01 g / L FeSO4·7H2O, 1 mg / L vitamin B1, 1 mg / L vitamin B6, 0.025 mg / L biotin, and 4 mg / L vitamin B12.

[0066] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A RhtA gene promoter, comprising a nucleotide sequence in which the -268th base of the nucleotide sequence shown in SEQ ID NO:3 is mutated from thymine to cytosine, and the nucleotide sequence of the promoter is shown in SEQ ID NO:

4.

2. A mutant of RhtA, wherein the mutation site comprises at least one of mutation point a and mutation point b; Mutation point a: The 43rd position of the amino acid sequence shown in SEQ ID NO.1 is mutated from valine V to alanine A; Mutation point b: The 271st position of the amino acid sequence shown in SEQ ID NO.1 mutates from alanine A to valine V.

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

4. The expression cassette according to claim 3, characterized in that A gene encoding the promoter according to claim 1 and the mutant of RhtA according to claim 2, wherein the mutant of RhtA comprises mutation site a.

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

6. The recombinant vector according to claim 5, characterized in that The promoter nucleotide sequence shown in SEQ ID NO: and / or the nucleotide sequence of the RhtA mutant according to claim 2 are introduced into a plasmid to construct the construct.

7. A recombinant strain comprising the recombinant vector according to claim 6.

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

9. Use of the promoter according to claim 1 in increasing L-homoserine production or preparing L-homoserine.

10. Use of the recombinant strain according to claim 7 or 8 in increasing the yield of L-homoserine or preparing L-homoserine.

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