CN113174356A

CN113174356A - Recombinant bacterium for producing threonine and application thereof

Info

Publication number: CN113174356A
Application number: CN202110501354.4A
Authority: CN
Inventors: 刘慧敏; 尹春筱; 康培
Original assignee: Langfang Meihua Bio Technology Development Co Ltd
Current assignee: Xinjiang Meihua Amino Acid Co ltd
Priority date: 2021-05-08
Filing date: 2021-05-08
Publication date: 2021-07-27
Anticipated expiration: 2041-05-08
Also published as: CN113174356B

Abstract

The invention relates to the technical field of genetic engineering, in particular to a recombinant bacterium for producing threonine and application thereof. The recombinant bacterium has an ability to produce L-threonine, and the copy number of yaaA gene in the recombinant bacterium is not less than 2. According to the invention, the expression level of yaaA protein in bacteria is effectively improved by improving copy of yaaA gene in bacterial genome, so that the L-threonine production capability of bacteria is obviously improved. The production efficiency of bacterial L-threonine can be effectively improved by improving the expression level of yaaA gene, and the method has important significance in the field of L-threonine production.

Description

Recombinant bacterium for producing threonine and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a recombinant bacterium for producing threonine and application thereof.

Background

L-threonine is one of 8 amino acids essential for human and animal growth, and is widely used in feed, food additives, preparation of auxiliary materials for medicines, and the like. Currently, L-threonine is mainly produced by fermentation of microorganisms, and various bacteria are available for L-threonine production, such as mutant strains induced by wild-type strains of Escherichia coli, Corynebacterium, Serratia, and the like, as production strains. Specific examples include amino acid analogue-resistant mutants or various auxotrophs such as methionine, lysine, isoleucine and the like (Japanese patent application laid-open No. 224684/83; Korean patent application laid-open No. 8022/87). However, in the conventional mutation breeding, the strain grows slowly and generates more byproducts due to random mutation, so that a high-yield strain is not easy to obtain.

With the increasing demand of threonine, the construction and modification of high-yield threonine strains are particularly important. In the Chinese patent CN03811059.8, the threonine synthesis key gene thrABC expression is enhanced by utilizing Escherichia coli and deleting 39bp sequences from the-56 th to-18 th positions of the threonine operon sequence, and the threonine productivity is improved by 22%. Kwang Ho Lee (Kwang Ho Lee et al, Systems metabolism engineering for L-threonine production, Mol Syst biol.2007; 3:149) and the like utilize a system metabolic engineering strategy, the feedback inhibition of products is relieved by mutating the genes thrA and lysC for encoding aspartate kinase I and III, byproducts glycine and isoleucine are removed by knocking out tdh and weakening ilvA, more precursors and the like are provided for threonine synthesis by inactivating competitive pathway genes metA and lysA, and the finally obtained TH28C (pBRThrABCR3) strain can produce 82.4g/L of acid after being fermented for 50h, and the conversion rate of the acid is 39.3%. In Chinese patent 201611250306.8, MHZ-0215-2 strain, which has threonine production of 12.4g/L, conversion rate of about 16.2% and no plasmid load, was obtained by enhancing pntAB gene and introducing pyc gene heterologously. In Chinese patent 202011388854.3, the biotin-enriched strain MHZ-0217-4(IS1:: bioABFCD, birA G57S) constructed from MHZ-0215-2 strain had a yield of 20.2G/l, a conversion rate of 23.8%, and no plasmid load.

Disclosure of Invention

In order to solve the problems of the prior art, the present invention provides a recombinant bacterium for producing threonine and use thereof.

In a first aspect, the present invention provides a threonine-producing recombinant bacterium having an ability to produce L-threonine, in which the copy number of yaaA gene is not less than 2.

Further, the recombinant bacterium is one or more of escherichia coli, corynebacterium or serratia, and preferably escherichia coli.

Further, the starting strain of the recombinant bacterium is MHZ-0215-2 or MHZ-0217-4.

Further, the yaaA gene includes a nucleotide sequence shown as SEQ ID NO. 1.

The invention further provides the use of the recombinant bacterium for the production of L-threonine.

In a second aspect, the present invention provides a method for producing L-threonine, comprising:

producing L-threonine by the recombinant bacterium.

Further, performing fermentation culture on the recombinant bacteria; the fermentation medium comprises the following components:

75-100 g/L of glucose, 5-10 g/L of corn steep liquor, 5-12 g/L of soybean meal hydrolysate and 0.4-0.6 g/L, KH g/L of magnesium sulfate heptahydrate₂PO₄ 0.5～1.5g/L、FeSO₄ 20～40mg/L、MnSO₄20-40 mg/L, 8-12 g/L aspartic acid, 40-60 mu g/L biotin and 400-600 mu g/L thiamine.

Further, the conditions of the fermentation culture are as follows:

fermenting and culturing at 36-38 ℃ and 80-120 rpm.

In a third aspect, the present invention provides a method for increasing the ability of a bacterium to produce L-threonine, comprising:

increasing the expression level of yaaA gene in the genome of said bacterium.

Further, the bacterium is one or more of escherichia coli, corynebacterium or serratia, preferably escherichia coli.

The invention has the following beneficial effects:

according to the invention, the copy number of yaaA genes in bacteria is increased, the yaaA genes in the bacteria are overexpressed, the expression level of yaaA protein is increased, the saccharic acid conversion efficiency of the starting bacteria and the yield of L-threonine production are obviously increased, and the L-threonine yield is increased by 12.97-15.45%.

The method provided by the invention can effectively improve the yield of L-threonine produced by bacteria, and has important significance in the field of L-threonine production.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

The invention respectively uses MHZ-0215-2 and MHZ-0217-4 as starting strains (the MHZ-0215-2 strain is disclosed in Chinese patent 201611250306.8, and the MHZ-0217-4 strain is disclosed in Chinese patent 202011388854.3), and related transformation is carried out on the genome of the starting strains to strengthen the expression of yaaA genes of the strains. The main mode is to increase the copy number of yaaA gene, thereby improving the expression amount of yaaA protein.

The CRISPR-Cas9 gene Editing technology (Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al.

In the following examples, Kanamycin (Kanamycin) was used at a final concentration of 50. mu.g/mL in the medium, and spectinomycin (spectinomycin) was used at a final concentration of 50. mu.g/mL in the medium.

In the following examples, all reagents used are commercially available. Parental strains of threonine-producing strains with high conversion rates provided by the invention are MHZ-0215-2 and MHZ-0217-4, and belong to W3110 (Escherichia).

The sequences of the primers used in the following examples are shown in the following table:

table 1 primer sequences used in the examples

The names of the genes referred to in the following examples are explained below:

yaaA: peroxide anti-stress protein;

a bioA: ademetionine-8-amino-7-oxononanoic acid aminotransferase;

and (3) bioB: a biotin synthase;

and (3) bioF: 8-amino-7-oxononanoic acid synthase;

and (3) bioC: malonyl carrier protein methyltransferase;

and (3) bioD: a desthiobiotin synthase;

and (3) birA: DNA binding transcription repressor/biotin- [ acetyl-CoA-carboxylase ] ligase;

pyc: a pyruvate carboxylase.

Example 1 preparation of a YaaA Gene-potentiated Strain MHZ-0218-1 (two copies)

1. pTargetF-N20(yaaA2) plasmid and Donor DNA construction

(1) pTargetF plasmid is used as a template (from Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. appl. environ Microbiol,2015), pTF-sgRNA-F/pTF-sgRNA-R primer pair is selected, pTF linear plasmid with N20 is amplified, the linear plasmid is assembled at 37 ℃ by using a seamless assembly ClonExpress kit, then Trans1-T1 competent cells are transformed, and pTargetF-N20(yaaA2) is obtained and subjected to PCR identification and sequencing verification.

(2) The upstream homology arm (r) is amplified by using the W3110 genome as a template and using yaaA2-UF/yaaA2-UR primer pair.

(3) yaaA gene 2 was amplified using the W3110 genome as a template and yaaA2-F/yaaA2-R primer set.

(4) The downstream homology arm (c) is amplified by using the W3110 genome as template and yaaA2-DF/yaaA2-DR primer pair.

(5) Using the first, the second and the third as templates, and selecting yaaA2-UF/yaaA2-DR primer pair to amplify up-yaaA-down full-length fragment, also called Donor DNA.

2. Competent cell preparation and electrotransformation

(1) The pCas plasmid (derived from Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. appl. environ Microbiol,2015) was electroporated into MHZ-0215-2 competent cells (see molecular clone III for both transformation and competent preparation).

(2) A single MHZ-0215-2(pCas) colony was picked up and cultured in 5mL LB tube containing kanamycin and arabinose at a final concentration of 10mM at 30 ℃ and 200r/min to OD₆₅₀After 0.4, electroporation competent cells were prepared (see molecular clone III).

(3) The pTargetF-N20(yaaA2) plasmid and the Donor DNA constructed in step 1 were simultaneously electroporated into MHZ-0215-2(pCas) competent cells (electroporation conditions: 2.5kV, 200. omega., 25. mu.F), spread on LB plates containing spectinomycin and kanamycin, and incubated at 30 ℃ until a single colony was visible.

3. Recombination verification

(1) Colony PCR verification was performed on the single colonies using primer pair yaaA2-F2/yaaA 2-R2.

(2) The amplification of the fragment of interest was performed with primer pair yaaA2-F2/yaaA2-R2, and the amplified product was sequenced to verify the integrity of the sequence.

4. Construction of related plasmid losses

(1) Single colonies with correct sequencing were picked and inoculated into 5mL LB tubes containing kanamycin and 0.5mM IPTG to a final concentration, incubated overnight at 30 ℃ and streaked onto LB plates containing kanamycin.

(2) Single colonies were picked and spotted on LB plates containing kanamycin and spectinomycin and LB plates containing kanamycin alone, and cultured overnight at 30 ℃ if they could not grow on LB plates containing kanamycin and spectinomycin and on LB plates containing kanamycin, indicating that the pTargetF-N20(yaaA2) plasmid had been lost.

(3) Positive colonies with lost pTargetF-N20(yaaA2) plasmid were picked, inoculated in non-resistant LB tubes, incubated at 42 ℃ for 8h, streaked onto LB plates, and incubated overnight at 37 ℃.

(4) Single colonies were picked as spots on both kanamycin-containing LB plates and on non-resistant LB plates, and if they failed to grow on kanamycin-containing LB plates, they showed loss of pCas plasmid, resulting in MHZ-0218-1(yaaA two-copy) strain.

Example 2 preparation of a YaaA Gene-potentiated Strain MHZ-0218-2 (two copies)

1. pTargetF-N20(yaaA2) plasmid and Donor DNA construction

2. Competent cell preparation and electrotransformation

(1) The pCas plasmid (derived from Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. appl. environ Microbiol,2015) was electroporated into MHZ-0217-4 competent cells (see molecular clone III for both transformation and competent preparation methods).

(2) A single MHZ-0217-4(pCas) colony was picked up and cultured in 5mL LB tube containing kanamycin and arabinose at a final concentration of 10mM at 30 ℃ and 200r/min to OD₆₅₀After 0.4, electroporation competent cells were prepared (see molecular clone III).

(3) The pTargetF-N20(yaaA2) plasmid and the Donor DNA constructed in step 1 were simultaneously electroporated into MHZ-0217-4(pCas) competent cells (electroporation conditions: 2.5kV, 200. omega., 25. mu.F), spread on LB plates containing spectinomycin and kanamycin, and incubated at 30 ℃ until a single colony was visible.

3. Recombination verification

4. Construction of related plasmid losses

(4) Single colonies were picked as spots on both kanamycin-containing LB plates and on non-resistant LB plates, and if they failed to grow on kanamycin-containing LB plates, they showed loss of pCas plasmid, resulting in MHZ-0218-2(yaaA two-copy) strain.

The threonine-producing genetically modified strains obtained in examples 1-2 are shown in Table 2.

TABLE 2 genetically engineered bacteria constructed in examples 1 and 2 of the present invention

Example 3 verification of shake flask fermentation of L-threonine producing genetically engineered bacteria

1. Taking 4 strains of MHZ-0215-2, MHZ-0217-4, MHZ-0218-1 and MHZ-0218-2 from the frozen tube, streaking and activating on an LB plate, and culturing at 37 ℃ for 18-24 h.

2. The cells were scraped from the plate in a ring and inoculated into a shake flask containing 50mL of seed medium (see Table 3) at 37 ℃ and 90 rpmCulturing at rpm for about 5 hours to OD₆₅₀And controlling to be within 2.

3. Transferring 2mL of the seed solution into a shake flask containing 20mL of a fermentation medium (shown in Table 4), performing fermentation culture at 100rpm with a reciprocating shaking table at 37 ℃ until residual sugar is exhausted, and measuring OD of a sample after fermentation is finished₆₅₀And the content of L-threonine was measured by HPLC, and the amount of residual sugar was measured by biosensing. To ensure the reliability of the experiment, the shaking flasks were subjected to 3 replicates and the average results for acid production and conversion are shown in table 5.

TABLE 3 seed culture Medium (g/L)

TABLE 4 fermentation Medium (g/L)

Composition (I)	Concentration of
		Glucose	85
Corn steep liquor	6
		Soybean meal hydrolysate	7.7
Magnesium sulfate heptahydrate	0.5
		KH2PO4	1.0
Aspartic acid	10
		FeSO4、MnSO4	30mg/L
Biotin	50μg
		Thiamine	500μg
pH	7.2

TABLE 5 comparison of productivity of threonine-producing genetically engineered bacteria

Note: denotes P value <0.01, indicating a clear difference from the control

As can be seen from Table 5, the yields of the L-threonine of the recombinant Escherichia coli prepared by the invention are higher than those of respective control strains, wherein the yield of the modified strain MHZ-0218-1 threonine is 15.59g/L, the shake flask conversion rate is 18.34%, the yield is 12.97% higher than that of the original strain, and the conversion rate is 13.21%; the yield of the modified strain MHZ-0218-2 threonine is 23.32g/L, the shake flask conversion rate is 27.44%, the yield is increased by 15.45% compared with that of the original strain, and the conversion rate is increased by 15.29%. The shake flask result can show that the threonine production capacity can be obviously improved by improving the expression quantity of the yaaA protein of the coliform strain.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> Gallery plum blossom Biotechnology development Co., Ltd

<120> a recombinant bacterium for producing threonine and use thereof

<130> KHP211113325.9

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 777

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgctgattc ttatttcacc tgcgaaaacg cttgattacc aaagcccgtt gaccaccacg 60

cgctatacgc tgccggagct gttagacaat tcccagcagt tgatccatga ggcgcggaaa 120

ctgacgcctc cgcagattag cacgctgatg cgcatcagcg acaaactggc gggtatcaac 180

gccgctcgct ttcatgactg gcagccagat ttcacgccgg cgaatgcccg ccaggcgatt 240

ctggcgttta aaggtgatgt ctacaccggc ttgcaggccg aaaccttcag cgaagacgat 300

ttcgattttg cccaacagca tttgcgaatg ctttccggct tgtatggcgt actccgcccg 360

ctcgatttaa tgcagcctta tcgtctggaa atggggatcc gtcttgagaa tgcccgaggg 420

aaagatctgt atcaattctg gggagatatc atcaccaaca agctgaacga ggcgctcgca 480

gcacaaggcg ataatgtggt gattaatctg gcgtcagatg agtattttaa atcggtgaag 540

ccgaagaaat tgaatgccga gattatcaag ccggtgttcc tcgatgagaa gaacggcaaa 600

tttaagatca tcagcttcta cgctaagaaa gcacgcggtc tgatgagtcg tttcattatt 660

gaaaatcggc tgaccaaacc agagcaactg actggtttta atagcgaagg ttacttcttt 720

gatgaagatt cctccagcaa tggcgaactg gtgtttaaac gctacgagca gcgttaa 777

<210> 2

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

agtaaatccg aaacagtctc gccgttttag agctagaaat agcaa 45

<210> 3

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ggcgagactg tttcggattt actagtatta tacctaggac tgagct 46

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

aaatccgaaa cagtctcgcc 20

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

aaatccgaaa cagtctcgcc 20

<210> 6

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

tgaaccacag gcgagagcag gcctggcgat aaccgtcttg tcggc 45

<210> 7

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

caagacggtt atcgccaggc ctgctctcgc ctgtggttca tacca 45

<210> 8

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cagaaatccg aaacagtctc ttaacgctgc tcgtagcgtt taaac 45

<210> 9

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

aacgctacga gcagcgttaa gagactgttt cggatttctg acact 45

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gttgtgccag agaacattcg 20

<210> 11

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gcagggtcag gttacagttg 20

<210> 12

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

atgccatagg cgatgagcaa 20

Claims

1. A recombinant bacterium which produces threonine and has an ability to produce L-threonine, wherein the copy number of yaaA gene in the recombinant bacterium is not less than 2.

2. The recombinant bacterium of claim 1, wherein the recombinant bacterium is one or more of escherichia coli, corynebacterium, or serratia, preferably escherichia coli.

3. The recombinant bacterium of claim 1, wherein the starting strain of the recombinant bacterium is MHZ-0215-2 or MHZ-0217-4.

4. The recombinant bacterium of claim 3, wherein yaaA gene comprises a nucleotide sequence as set forth in SEQ ID NO. 1.

5. Use of the recombinant bacterium of any one of claims 1-4 for the production of L-threonine.

6. A method for producing L-threonine, comprising:

l-threonine is produced by the recombinant bacterium according to any one of claims 1 to 4.

7. The method of claim 6, comprising:

subjecting the recombinant bacterium of any one of claims 1-4 to a fermentation culture; the fermentation medium comprises the following components:

8. The method according to claim 6 or 7, wherein the conditions of the fermentation culture are:

fermenting and culturing at 36-38 ℃ and 80-120 rpm.

9. A method for increasing the ability of a bacterium to produce L-threonine, comprising:

increasing the expression level of yaaA gene in the genome of said bacterium.

10. The method according to claim 9, wherein the bacteria is one or more of escherichia coli, corynebacterium or serratia, preferably escherichia coli.