CN119120333A

CN119120333A - A recombinant microorganism and its application in producing L-isoleucine

Info

Publication number: CN119120333A
Application number: CN202411066421.4A
Authority: CN
Inventors: 陈振; 刘洋
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2024-08-05
Filing date: 2024-08-05
Publication date: 2024-12-13

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses a recombinant microorganism and application thereof in the production of L-isoleucine. Compared with a starting strain, the recombinant microorganism expresses cimA mutants and ilvIH mutants, wherein the starting strain is escherichia coli or corynebacterium glutamicum, the amino acid sequence of the cimA mutants is shown as SEQ ID No.1, and the nucleotide sequence of the ilvIH mutants is shown as SEQ ID No. 5. L-isoleucine can be produced by fermentation using the recombinant microorganism of the present invention.

Description

Recombinant microorganism and application thereof in production of L-isoleucine

Technical Field

The invention relates to the technical field of microorganisms, in particular to a recombinant microorganism and application thereof in the production of L-isoleucine.

Background

L-isoleucine is one of eight essential amino acids, and is commonly referred to as a "branched chain amino acid" with L-valine and L-leucine because of the branched methyl group. L-isoleucine is an indispensable component in the physiological metabolism of animals and plants as a supplement to synthetic protein raw materials, and is widely used in medical treatment, feed, pharmacy, food and other fields. The market demand for L-isoleucine is expanding. The current industrialized production method of L-isoleucine mainly comprises an extraction method, a fermentation method and the like. The traditional chemical synthesis method has been eliminated because of the problems of complex reaction, low yield and the like. The extraction method needs strong acid to hydrolyze animal and plant protein tissues, which is easy to cause environmental pollution. Compared with the extraction method, the fermentation method is widely applied because of the advantages of simple operation, low cost, environmental friendliness, green sustainable development and the like.

In the microorganism, L-isoleucine is formed by amino transfer reaction of oxaloacetic acid, an intermediate metabolite of TCA cycle, to produce aspartic acid, and the aspartic acid is synthesized into a precursor substance L-threonine by 5-step enzyme catalytic reaction. L-threonine generates 2-ketobutyric acid by L-threonine dehydratase (ilvA code), L-isoleucine is synthesized from 2-ketobutyric acid by 4-step enzyme reaction, and finally the enzymes participating in the reaction in the last 4 steps are acetohydroxy acid synthase (ilvIH code), acetohydroxy acid isomerase reductase (ilvC code), dihydroxydehydratase (ilvD code) and branched-chain amino acid transaminase (ilvE code) in sequence. Currently, strategies to enhance isoleucine synthesis have focused mainly on enhancing the precursor threonine and threonine to isoleucine bioconversion processes. Further research is still needed on how to promote the synthesis of isoleucine.

Disclosure of Invention

It is an object of the present invention to provide a novel process for the fermentative production of L-isoleucine.

The invention provides a recombinant microorganism, which is compared with a starting strain, the recombinant microorganism expresses cimA mutant and ilvIH mutant, and overexpresses leuBCD, wherein the starting strain is escherichia coli or corynebacterium glutamicum, the amino acid sequence of the cimA mutant is shown as SEQ ID No.1, and the nucleotide sequence of the ilvIH mutant is shown as SEQ ID No. 5.

The invention makes a great deal of researches on how to improve the supply of metabolic precursor 2-ketobutyric acid (key precursor for synthesizing isoleucine) in microorganisms, and discovers that a novel way for producing L-isoleucine is obtained by introducing Methanocaldococcus jannaschii source citrate malate synthase mutant (cimA) and ilvIH mutant for relieving feedback inhibition of L-isoleucine. Furthermore, the invention provides a novel method for supplying the L-isoleucine metabolic precursor 2-ketobutyric acid. The introduction of exogenous mutants of citramalate synthase (cimA, EC 2.3.1.182) into microorganisms such as E.coli or C.glutamicum catalyzes the production of both acetyl-CoA and pyruvate to citramalate, which can be further converted to 2-ketobutyrate by expression of the leuBCD gene of the strain itself. The method for synthesizing the 2-ketobutyric acid can bypass the complex metabolic regulation of the strain, improve the synthesis efficiency of the 2-ketobutyric acid and the ratio of intracellular 2-ketobutyric acid to pyruvic acid, thereby further improving the yield of isoleucine on the basis of the prior art.

Preferably, the recombinant microorganism of the invention is further overexpressed leuBCD compared to the starting strain to better convert citramalate to 2-ketobutyrate.

More preferably, the recombinant microorganism of the invention further overexpresses thrABC and expresses ilvA mutant while expressing cimA mutant and ilvIH mutant, overexpressing leuBCD.

At this time, the recombinant microorganism can synthesize 2-ketobutyrate and L-isoleucine through both threonine pathway and citramalate synthase pathway, and both pathways synergistically enhance the yield of L-isoleucine.

In the recombinant microorganism, the nucleotide sequence of the cimA mutant is shown as SEQ ID No.2, the nucleotide sequence of leuBCD is shown as SEQ ID No.3, and the nucleotide sequence of thrABC is shown as SEQ ID No. 31.

The invention also provides application of the recombinant microorganism in fermentation production of L-isoleucine, genetic breeding of the microorganism for producing L-isoleucine or improvement of the yield of L-isoleucine synthesized by a biological method.

The invention also provides cimA mutant, the amino acid sequence of which is shown as SEQ ID No. 1.

The invention also provides a DNA molecule, the nucleotide sequence of which is shown as SEQ ID No.2, 4 or 5.

The invention also provides application of the DNA molecule in constructing recombinant microorganisms, wherein the recombinant microorganisms can ferment to produce L-isoleucine, and the initial strain is escherichia coli or corynebacterium glutamicum.

The cimA mutant of the invention can be used for being combined with other genetic engineering to construct a novel 2-ketobutyrate synthesis pathway in recombinant bacteria.

The present invention also provides a method for producing L-isoleucine by fermentation, which comprises the step of culturing the recombinant microorganism described above.

The invention also provides a method for constructing a recombinant microorganism for producing L-isoleucine, which comprises the steps of enabling a starting strain to express cimA mutants and ilvIH mutants, or further over-express leuBCD, or further over-express thrABC and express ilvA mutants, wherein the starting strain is escherichia coli or corynebacterium glutamicum, the amino acid sequence of the cimA mutants is shown as SEQ ID No.1, the nucleotide sequence of the ilvIH mutants is shown as SEQ ID No.5, the nucleotide sequence of the ilvA mutants is shown as SEQ ID No.4, the nucleotide sequence of the leuBCD is shown as SEQ ID No.3, and the nucleotide sequence of the thrABC is shown as SEQ ID No. 31.

The invention has the advantages that:

the present invention provides a novel method for producing 2-ketobutyric acid and isoleucine in a microorganism, which bypasses the complex metabolic regulation of threonine pathway and can significantly improve the yield and productivity of L-isoleucine when it exists together with the known L-isoleucine synthesis pathway (threonine pathway).

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention.

The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents and the like used in the examples below, unless otherwise indicated, are all those available commercially or may be prepared by methods conventional in the art.

The specific implementation mode of the invention comprises (1) constructing recombinant plasmids pBbA K-cimA, pBbA1K-cimA-leuBCD, pTrc99a-ilvAIH and pTrc99a-ilvIH, wherein the amino acid sequence of cimA is shown as SEQ ID No.1, the nucleotide sequence of cimA is shown as SEQ ID No.2, the sequence of leuBCD is shown as SEQ ID No.3, the sequence of ilvA is shown as SEQ ID No.4, the sequence of ilvIH is shown as SEQ ID No.5, (2) introducing the recombinant plasmids into escherichia coli for constructing an L-isoleucine production strain, and (3) fermenting and culturing the strain to detect the growth of thalli and the yield of L-isoleucine. Possible embodiments are as follows, but are not limited to the following examples.

EXAMPLE 1 construction of recombinant plasmids pBbA K-cimA, pBbA1K-cimA-leuBCD, pTrc99a-ilvAIH, pTrc99a-ilvIH

In this example, a Methanocaldococcus jannaschii-derived cimA mutant (comprising the point mutation I47V-E114A-H126Q-T204G-L238S, the sequence of which is shown in SEQ ID No.1, the nucleotide sequence of which is shown in SEQ ID No. 2), an E.coli-derived leuBCD (the sequence of which is shown in SEQ ID No. 3), an E.coli-derived ilvA mutant (ilvA comprising the double point mutation F352A-R362F, the sequence of which is shown in SEQ ID No. 4) which releases the feedback inhibition by L-isoleucine, and an E.coli-derived ilvIH mutant (ilvI comprising the double point mutation G41A-C50T, the sequence of which is shown in SEQ ID No. 5) which releases the feedback inhibition by L-isoleucine were constructed on a vector.

Specifically, PCR was performed using the synthesized cimA gene as a template and cimA-F (TCTTTTAAGAAGGAGATATACATATGATGGTGCGCATTTTTGATACCAC, SEQ ID No. 6) and cimA-R2 (CCTTACTCGAGTTTGGATCCTTAGTTCAGCAGCATGTTAATGCCTTCCAT, SEQ ID No. 32) as primers, obtaining a gene fragment cimA of about 1500bp and performing PCR product purification. PCR was performed using plasmid pBbA K (from Addgene) as a template and primers pBbA K-F (GGATCCAAACTCGAGTAAGGATCTCCAGGCA, SEQ ID No.10 and pBbA K-R (ATGTATATCTCCTTCTTAAAAGATCTTTTGAATTCTGA, SEQ ID No. 11) to obtain gene fragment pBbA K of about 3500bp and purification of the PCR product, and fragments cimA and pBbA K were recombined by a recombination cloning kit (Vazyme, C115) and then introduced into host E.coli DH 5. Alpha. And the recombinant plasmids pBbA K-cimA were obtained by screening and extraction.

PCR was performed using the synthesized cimA gene as a template and cimA-F (TCTTTTAAGAAGGAGATATACATATGATGGTGCGCATTTTTGATACCAC, SEQ ID No. 6) and cimA-R (TTAGTTCAGCAGCATGTTAATGCCTTCCATCAC, SEQ ID No. 7) as primers, obtaining a gene fragment cimA of about 1500bp and performing PCR product purification. The genome of E.coli MG1655 ATCC 700926 was used as a template, and PCR was performed with primers leuB-F (AACATGCTGCTGAACTAATTTAAGAAGGAGATATACATATGTCGAAGAATTACCATATTGCCGTAT, SEQ ID No. 8) and leuD-R (AGATCCTTACTCGAGTTTGGATCCTTAATTCATAAACGCAGGTTGTTTTGCTTCATAAG, SEQ ID No. 9), obtaining a gene fragment leuBCD of about 3000bp and performing PCR product purification. PCR was performed using plasmid pBbA K (purchased from Addgene) as a template and primers pBbA K-F (GGATCCAAACTCGAGTAAGGATCTCCAGGCA, SEQ ID No.10 and pBbA1K-R (ATGTATATCTCCTTCTTAAAAGATCTTTTGAATTCTGA, SEQ ID No. 11) to obtain gene fragment pBbA K of about 3500bp and purification of PCR products, the fragments cimA, leuBCD and pBbA K were recombined by a recombination cloning kit (Vazyme, C115) and then introduced into host E.coli DH 5. Alpha. And the resultant recombinant plasmid pBbA K-cimA-leuBCD was obtained by screening and extraction.

The genome of Escherichia coli MG1655 was used as a template, PCR was performed with the primer ilvA-F（ggataacaatttcacacaggaaacagaccatggctgactcgcaacccctgtccggtg,SEQ ID No.12）、ilvA(F352A)-R（ccgccaagcagttggcagaatttgagcgcgctgcctttttcttccggaatgg,SEQ ID No.13） to obtain a gene fragment ilvA-1 of about 1000bp, and PCR product purification was performed. PCR was performed with primers ilvA (R362F) -F (TCAAATTCTGCCAACTGCTTGGCGGGTTCTCGGTCACCGAGTTCAACTACCGTTT, SEQ ID No. 14) and ilvA-R (ACCCGCCAAAAAGAACCTGAACGCCGGGTTATTGGTTTCGTCGTGGCAATCG, SEQ ID No. 15) to obtain gene fragment ilvA-2 of about 500bp and PCR product purification was performed. The genome of Escherichia coli MG1655 was used as a template, PCR was performed with a primer ilvI-F（cccggcgttcaggttctttttggcgggttagtttaagaaggagatatacatatggagatgttgtctggagccgaga,SEQ ID No.16）、ilvH(G41A,C50T)-R（ccaatcacgcggAataacgcgTctgattcattttcgagtaagactgat,SEQ ID No.17） to obtain a gene fragment ilvIH-1 of about 1800bp, and PCR product purification was performed. PCR was performed with primers ilvH (G41A, C50T) -F (CAGACGCGTTATTCCGCGTGATTGGCCTTTTTTCCCAG, SEQ ID No. 18) and ilvH-R (CAGGTCGACTCTAGAGGATCCTCAACGCATTATTTTATCGCCGCGCGA, SEQ ID No. 19) to obtain gene fragment ilvIH-2 of about 400bp and PCR product purification was performed. The recombinant plasmid pTrc99a-ilvAIH is obtained by carrying out PCR with the plasmid pTrc99a as a template and the primers pTrc99a-F (GGATCCTCTAGAGTCGACCTGCAGGCATGC, SEQ ID No.20 and pTrc99a-R (GGTCTGTTTCCTGTGTGAAATTGTTATCCG, SEQ ID No. 21) to obtain the gene fragment pTrc99a of about 4000bp and purifying the PCR product, and recombining the fragments ilvA-1, ilvA-2, ilvIH-1, ilvIH-2 and pTrc99a by a recombination cloning kit (Vazyme, C115) and then introducing the recombinant plasmid into host E.coli DH 5a, and screening and extracting.

PCR was performed using the recombinant plasmid pTrc99a-ilvAIH as a template and primers ilvI-F2 (TAACAATTTCACACAGGAAACAGACCATGGAGATGTTGTCTGGAGCCGAGATG, SEQ ID No. 22) and ilvH-R (SEQ ID No. 19) to obtain a gene fragment ilvIH of about 2200bp and to purify the PCR product. PCR was performed using the plasmid pTrc99a as a template and the primers pTrc99a-F (SEQ ID No. 20) and pTrc99a-R (SEQ ID No. 21), obtaining the gene fragment pTrc99a of about 4000bp and performing PCR product purification. The fragments ilvIH and pTrc99a are recombined by a recombination cloning kit (Vazyme, C115), and then introduced into a host E.coli DH5 alpha, and the recombinant plasmid pTrc99a-ilvIH is obtained by screening and extracting.

EXAMPLE 2 construction of L-isoleucine-producing engineering bacterium

PCR was performed using the genome of E.coli MG1655 as a template and primers tdh-UP-F (TTCCTGCTTTGATGCTAACGGTGGCCT, SEQ ID No. 23) and tdh-UP-R (GCATTATACGAGCCGGATGATTAATTGTCAAAGTCCCGCAGATGGCTGTTTTAC, SEQ ID No. 24) as primers, to obtain a gene fragment tdh-UP of about 1000bp and to purify the PCR product. PCR was performed using the genome of E.coli MG1655 as a template and primers tdh-DOWN-F (GATGATGAATCATCAGTAACACGAACAAGGGCTGGTATTCCA, SEQ ID No. 25) and tdh-DOWN-R (GGCATAATTTCGATTTAATTTCTC, SEQ ID No. 26) as primers, to obtain a gene fragment tdh-DOWN of about 1000bp and to purify the PCR product. The genome of Escherichia coli MG1655 is used as a template, primers thrABC-F (ATCCGGCTCGTATAATGCACACAGGAAACAGACCATGCGAGTGTTGAAGTTCGGCGGTA, SEQ ID No. 27) and thrABC-R (TGGAATACCAGCCCTTGTTCGTGTTACTGATGATTCATCATCAATTTAC, SEQ ID No. 28) are used as primers for PCR, a gene fragment thrABC of about 1000bp is obtained, and PCR product purification is carried out.

The fragments tdh-UP, tdh-DOWN and thrABC were subjected to overlap PCR to obtain targeting fragments. The plasmid pTarget（Jiang, Y., Chen, B., Duan, C.L., Sun, B.B., Yang, J.J., and Yang, S. (2015) Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol 81: 2506–2514.Jiang,et al. 2015） was used as a template, and primers tdh-N20-F (TCGTCTGATATGTCTATCGACGTTTTAGAGCTAGAAATAGCAAGTTAAAAT, SEQ ID No. 29) and tdh-N20-R (GTCGATAGACATATCAGACGACTAGTATTATACCTAGGACTGAGCTAG, SEQ ID No. 30) were used for amplification to obtain pTarget-tdh. The targeting fragment is transferred into escherichia coli MG1655 by electrotransformation by an electroporation apparatus (Berle), the electric shock condition is that the voltage is 2.5KV, the resistance is 200 Ω, the capacitance is 25 muF (the width of an electric shock cup is 2 mm), and recombinant bacteria capable of producing threonine are obtained by screening, and the strain is named as E.coil-Deltatdh:: ptrc-thrABC.

The recombinant plasmids pTrc99a-ilvAIH and pBbA K prepared in example 1 were transformed by electrotransformation (conditions as above) into E.coil-Deltatdh:: ptrc-thrABC, and the obtained recombinant strain was named E.coil-Deltatdh::: ptrc-thrABC/ilvAIH, and was able to synthesize L-isoleucine via the threonine pathway without the citramalate synthase pathway.

The recombinant plasmids pTrc99a-ilvIH and pBbA K-cimA-leuBCD prepared in example 1 were transferred to E.coil-MG 1655 by electrotransformation (conditions as above), the obtained recombinant strain was named E.coil-MG 1655/ilvIH-cimA-BCD, the recombinant plasmids pTrc99a-ilvIH and pBbA K-cimA were transferred to E.coil-MG 1655 by electrotransformation (conditions as above), and the obtained recombinant strain was named E.coil-MG 1655/ilvIH-cimA, which were able to synthesize 2-ketobutyric acid and L-isoleucine only by the citramalate synthase pathway since threonine synthesis pathway key gene thrABC and ilvA releasing feedback inhibition by L-isoleucine were not overexpressed. Recombinant plasmids pTrc99a-ilvIH and pBbA K were transferred to E.coil-MG 1655 by electrotransformation (conditions as above), and the obtained recombinant strain was designated as E.coil-MG 1655/ilvIH, and as a control group, the strain was overexpressed only ilvIH gene and 2-ketobutyrate could not be synthesized efficiently.

The recombinant plasmids pTrc99a-ilvAIH and pBbA K-cimA-leuBCD prepared in example 1 were transformed by electrotransformation (conditions as above) into E.coil-Deltatdh:: ptrc-thrABC, and the obtained recombinant strain was named E.coil-Deltatdh::: ptrc-thrABC/ilvAIH-cimA, which was capable of synthesizing 2-ketobutyric acid and L-isoleucine simultaneously via the threonine pathway and the citramalate synthase pathway.

EXAMPLE 3 production of L-isoleucine by recombinant E.coli fermentation culture

Recombinant strains E.coil- MG1655/ilvIH,E.coil- MG1655/ilvIH- cimA,E.coil- MG1655/ilvIH- cimA-BCD,E.coil-△tdh::Ptrc-thrABC/ilvAIH and E.coil-. DELTA.tdh prepared in example 2: ptrc-thrABC/ilvAIH-cimA were cultured overnight on LB plates. From the fresh plate, single colony inoculation containing 5ml LB medium test tube, 37 degrees C,200rpm culture for 12 hours.

Inoculated in 500ml baffle shake flasks containing 50 ml% of fermentation medium at 37℃and 200rpm to OD ₆₀₀ of 0.6, added with 0.1mM IPTG and co-cultured for 48h.

Each liter of fermentation medium comprises 20g of glucose, 0.8g of magnesium sulfate heptahydrate, 4g of diamine hydrogen phosphate, 6.67g of monopotassium phosphate, 1.35g of potassium citrate, 20.9g of 3-morpholinopropane sulfonic acid, 2.5g of yeast powder, 50mg of ferrous sulfate heptahydrate, 10mg of calcium chloride dihydrate, 11mg of zinc sulfate heptahydrate, 2.5mg of manganese sulfate tetrahydrate, 5mg of copper sulfate pentahydrate, 0.5mg of ammonium molybdate and 0.1mg of sodium borate decahydrate.

The concentration of the product and the growth of the strain were measured by liquid chromatography during fermentation, and the results are shown in tables 1 and 2. As can be seen from tables 1 and 2, the control strain E.coil-MG 1655/ilvIH was unable to synthesize isoleucine, and L-isoleucine was synthesized in this strain by the citral synthase pathway alone after introducing cimA (strain E.coil-MG 1655/ilvIH-cimA) or cimA-leuBCD (strain E.coil-MG 1655/ilvIH-cimA-BCD). Meanwhile, after cimA-leuBCD is further introduced into the strain E.coil-Deltatdh:: ptrc-thrABC/ilvAIH which retains the natural threonine and isoleucine synthesis pathways of escherichia coli (the strain E.coil-Deltatdh::: ptrc-thrABC/ilvAIH-cimA), the yield and production efficiency of L-isoleucine can be obviously improved.

TABLE 1 growth of different strains (OD ₆₀₀)

TABLE 2L-isoleucine production cases (g/L) for different strains

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims

1. A recombinant microorganism is characterized in that the recombinant microorganism expresses cimA mutant and ilvIH mutant compared with a starting strain, wherein the starting strain is escherichia coli or corynebacterium glutamicum;

The amino acid sequence of the cimA mutant is shown as SEQ ID No.1, and the nucleotide sequence of the ilvIH mutant is shown as SEQ ID No. 5.

2. The recombinant microorganism according to claim 1, wherein the recombinant microorganism is further overexpressed leuBCD compared to the starting strain.

3. The recombinant microorganism according to claim 2, wherein the recombinant microorganism further overexpresses thrABC and expresses an ilvA mutant compared to the starting strain, the nucleotide sequence of the ilvA mutant being shown in SEQ ID No. 4.

4. The recombinant microorganism according to claim 3,

The nucleotide sequence of the cimA mutant is shown as SEQ ID No. 2;

the nucleotide sequence of leuBCD is shown as SEQ ID No. 3;

the nucleotide sequence of thrABC is shown as SEQ ID No. 31.

5. Use of a recombinant microorganism according to any one of claims 1-4 for the fermentative production of L-isoleucine, genetic breeding of a microorganism for the production of L-isoleucine or for increasing the yield of L-isoleucine synthesized by a biological method.

6. A cimA mutant is characterized in that the amino acid sequence is shown as SEQ ID No. 1.

7. A DNA molecule, wherein the nucleotide sequence is shown in SEQ ID No.2, 4 or 5.

8. Use of a DNA molecule according to claim 7 for the construction of a recombinant microorganism which can ferment to produce L-isoleucine, the starting strain being escherichia coli or corynebacterium glutamicum.

9. A process for producing L-isoleucine by fermentation, which comprises the step of culturing the recombinant microorganism as claimed in any one of claims 1 to 4.

10. A method for constructing a recombinant microorganism for producing L-isoleucine is characterized by comprising the steps of enabling a starting strain to express cimA mutants and ilvIH mutants, or further over-express leuBCD, or further over-express thrABC and express ilvA mutants, wherein the starting strain is escherichia coli or corynebacterium glutamicum, the amino acid sequence of the cimA mutants is shown as SEQ ID No.1, the nucleotide sequence of the ilvIH mutants is shown as SEQ ID No.5, the nucleotide sequence of the ilvA mutants is shown as SEQ ID No.4, the nucleotide sequence of the leuBCD is shown as SEQ ID No.3, and the nucleotide sequence of the thrABC is shown as SEQ ID No. 31.