CN112662573B - Microbial strain for efficiently synthesizing L-piperazinic acid and construction method and application thereof - Google Patents

Abstract

The invention relates to the technical field of genetic engineering, and discloses a microbial strain for efficiently synthesizing L-piperazinic acid, a construction method and application thereof. The microbial strain provided by the invention is used for further transforming a vector containing an L-piperazines synthetase encoding gene ktzT or hmtC in a aureobasidium pullulans D-OLCF strain for producing melanin, or integrating the L-piperazines synthetase encoding gene ktzT or hmtC into a genome of the aureobasidium pullulans D-OLCF strain for producing melanin. The invention selects an appropriate source of L-piperazine acid synthetase gene, constructs a microbial strain with high L-piperazine acid yield, and can further promote the L-piperazine acid yield by introducing the inhibition of iron ions to be removed by the L-ornithine hydroxylase coding gene SidA; the L-piperazine acid is directly produced by converting the fermentation raw material by the microbial strain provided by the invention, the synthesis process does not need to depend on a chemically synthesized platform compound, the reaction condition is mild and easy to control, and the method is environment-friendly.

Description

Microbial strain for efficiently synthesizing L-piperazinic acid and construction method and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a microbial strain for efficiently synthesizing L-piperazinic acid and a construction method and application thereof.

Background

Piperazine acid is a unique non-protein amino acid containing a nitrogen-nitrogen (N-N) bond. Piperazinoic acid is present in many natural products with significant biological activity, such as the natural immunosuppressant sanglifehrin (a) and the antitumor active peptide luzoneptins (luzopeptins). Piperazine acid is a first-line antihypertensive drug cilazapril

A synthetic precursor. Furthermore, the piperazine acid has a structurally rigid conformation, which makes it possible to introduce, as building blocks, β -turn structures in peptide synthesis. These unique properties have motivated synthetic chemists to devote their efforts to the development of chemical synthetic pathways for piperazine acids as important intermediates in the synthesis of drugs and chemicals. Thus, the compound was investigatedThe high-efficiency synthesis of the compound has important and wide significance.

Over the last few years, the pure stereoselective specific synthesis of piperazinic acids and the piperazinic acid building blocks within the macromolecular chemical building blocks have received increasing attention. The piperazinic acid can be synthesized by two methods, wherein one method takes a chiral compound as an initial raw material, such as L- (+) glutamic acid, and the compound is obtained by nine-step synthesis, and the method has the advantages of long line, high cost, complex process, high reaction difficulty and lower total yield (less than 30%); the second method is to use an achiral compound as a raw material and perform Diels-Alder reaction, although the process is relatively simple and the yield is high, the obtained product is racemic and needs to be subjected to chemical resolution, and common chemical resolving agents are expensive.

The biotransformation method can overcome the defects, the reaction of biotransformation of piperazine acid directly converts the fermentation raw material to produce the compound, the synthesis process does not need to depend on a chemically synthesized platform compound, the reaction condition is mild and easy to control, and the method is environment-friendly. Therefore, how to efficiently synthesize the piperazine acid by a biological method is a key technical problem to be solved by the invention.

Disclosure of Invention

In view of the above, the present invention aims to provide a microbial strain for efficiently synthesizing L-piperazinic acid and a construction method thereof, such that the microbial strain can be used to obtain a very high yield of L-piperazinic acid;

the invention also aims to provide the application of the microbial strain in the fermentation production of L-piperazinic acid or the preparation of a microbial product for the fermentation production of L-piperazinic acid;

another object of the present invention is to provide a method for producing L-piperazinic acid by fermentation using the microorganism strain.

In order to achieve the above purpose, the invention provides the following technical scheme:

a microbial strain for efficiently synthesizing L-piperazinic acid is characterized in that a carrier containing an L-piperazinic acid synthetase encoding gene ktzT or hmtC is further transformed in an aureobasidium melanoidinum D-OLCF strain, or the L-piperazinic acid synthetase encoding gene ktzT or hmtC is integrated into a genome of the aureobasidium melanoidinum D-OLCF strain;

the aureobasidium pullulans D-OLCF (hereinafter referred to as DF1) is a compound which has a preservation number of CCTCC NO: knock-out of ornithine carbamoyl transferase gene OTC and L-ornithine-N on the basis of Aureobasidium melanogenin HN6.2 strain of M2020340 ⁵ Transacetylase gene sidL, FC synthetase gene sidC and L-ornithine-N ⁵ -a strain of the hydroxy-transacetylase gene sidF.

Preferably, the L-piperazine synthetase encoding gene ktzT is derived from Kutzneria sp.744; the gene hmtc encoding the L-piperazine synthetase is derived from Streptomyces himastatinicus ATCC 53653. In a specific embodiment of the present invention, the sequence of the coding gene ktzT of L-piperazinic acid synthetase derived from Kutzneria sp.744 is shown in SEQ ID NO. 1, and the coding gene hmtc of L-piperazinic acid synthetase derived from Streptomyces himastatinicus ATCC 53653 is shown in SEQ ID NO. 2.

In order to further improve the yield of the microbial strains, the method also comprises knocking out the original L-ornithine hydroxylase coding gene sidA in DF1 and converting a vector containing the L-ornithine hydroxylase coding gene SidA shown in SEQ ID NO. 8; or replacing the original L-ornithine hydroxylase coding gene sidA with the L-ornithine hydroxylase coding gene sidA shown in SEQ ID NO. 8 in the DF1 genome. When transforming a vector containing the L-ornithine hydroxylase encoding gene SidA, SidA can be introduced into the same vector as ktzT or hmtc for efficiency reasons.

In the concrete embodiment of the invention, the invention also provides L-piperazine synthetase encoding genes derived from Streptomyces flaveolus DSM9954, Streptomyces sp, Actinomanda atramentaria DSM 43919 and Pseudonocardia sp.HH130629-09, which are sfAC, padO, matF and XF36 in sequence, and the sequences are shown as SEQ ID NO:3-6 in sequence; the microbial strain has the L-piperazinic acid yield of more than 150mg/L, and has very obvious yield advantage compared with the microbial strain constructed by the genes.

Based on the above, the invention provides the application of the microbial strain in fermentation production of L-piperazinic acid or the application in preparation of a microbial product for fermentation production of L-piperazinic acid.

Meanwhile, the invention also provides a construction method of the microbial strain, which comprises the steps of constructing a vector containing the L-piperazinic acid synthetase encoding gene ktztT or hmtc, and then transforming the vector into DF1 to obtain the microbial strain. In addition, the coding gene ktztt or hmtc can be integrated directly into the DF1 genome by prior art methods, for example, by constructing upstream and downstream homology arms for integration by yeast homologous recombination.

In the construction process, the original gene sidA of L-ornithine hydroxylase in DF1 can be further knocked out and a vector containing the gene SidA of L-ornithine hydroxylase coding shown in SEQ ID NO. 8 can be transformed; or replacing the original L-ornithine hydroxylase coding gene sidA with the L-ornithine hydroxylase coding gene SidA shown in SEQ ID NO. 8 in the DF1 genome; in transforming vectors containing the L-ornithine hydroxylase encoding gene SidA, SidA can be introduced into the same vector as ktzT or hmtc for efficiency reasons.

In a specific embodiment of the invention, the vector is pAMEXloxp-1 plasmid.

In addition, the invention also provides a method for producing L-piperazinic acid by fermentation, namely inoculating the microbial strain to a piperazinic acid production medium to produce L-piperazinic acid by fermentation.

Preferably, the piperazine acid producing medium is a CM medium; in a specific embodiment of the invention, the CM medium is: 10-50 g/L glucose, 10-30g/L peptone, 1-5 g/L dipotassium hydrogen phosphate, 1-2 g/L citric acid monohydrate, 1-5 g/L YNB, 0.01-0.1 g/L magnesium sulfate, 0.001-0.005 g/L zinc sulfate, and pH 6-7.

Preferably, the fermentation is carried out at 28 ℃ and 180rpm for 120 h.

According to the technical scheme, the L-piperazinic acid synthetase gene with a proper source is selected, the microbial strain with high L-piperazinic acid yield is constructed, and the inhibition of iron ions can be further improved by introducing the coding gene SidA of the L-ornithine hydroxylase to decompose the iron ions; the L-piperazine acid is directly produced by converting the fermentation raw materials by the microbial strains provided by the invention, the synthesis process does not need to depend on a chemically synthesized platform compound, the reaction condition is mild and easy to control, and the method is environment-friendly. The method solves the problems of low stereoselectivity, serious pollution, complex process, high cost, low yield and the like caused by a chemical production method.

Biological material preservation information description

And (3) classification and naming: aureobasidium melanogenoum HN6.2, which has been deposited with the China center for type culture Collection on 23/7/2020, addresses: china, Wuhan university, preservation number CCTCC NO: m2020340.

Drawings

FIG. 1 shows a map of pAMEXloxp-1 vector;

FIG. 2 shows the determination of the absolute configuration of L-piperazinoic acid produced by the microorganism strain of the present invention; wherein A is an FDAA derivatization liquid phase diagram of an L-piperazinic acid standard product; b is an FDAA derivatization mass spectrogram of an L-piperazinic acid standard product; c is a liquid phase diagram of L-piperazine acid FDAA derivatization produced by the microbial strain; d is an FDAA derivatization mass spectrogram of L-piperazinic acid produced by the microbial strain;

FIG. 3 shows the production of L-piperazinoic acid by the microorganism strain of the present invention ¹ H NMR spectrum;

FIG. 4 shows the production of L-piperazinoic acid by the microorganism strain of the present invention ¹³ C NMR spectrum;

FIG. 5 shows a HMBC spectrum of L-piperazinoic acid produced by the microbial strain of the invention;

FIG. 6 shows HSQC spectra of L-piperazinic acid produced by the microbial strains of the present invention;

FIG. 7 shows the LC-MS detection results of L-piperazinoic acid Fmoc-Cl derivatization by the microbial strains of the present invention;

FIG. 8 shows the L-piperazinic acid production in iron deficiency medium and iron deficiency medium by microbial strains DFK1 and DFAK1 of the present invention.

Detailed Description

The invention discloses a microbial strain for efficiently synthesizing L-piperazine acid and a construction method and application thereof. It is expressly intended that all such similar substitutes and modifications apparent to those skilled in the art are deemed to be included in the invention. The microorganism strains and the construction methods and applications thereof have been described in the preferred embodiments, and it is obvious to those skilled in the art that the technology of the present invention can be implemented and applied by modifying or appropriately changing and combining the microorganism strains and the construction methods and applications thereof without departing from the content, spirit and scope of the present invention.

The nucleic acid fragment, primer probe, reagent and the like used in the present invention can be synthesized and purchased by a reagent company.

The main experimental materials used in the examples of the invention are as follows:

the HN6.2 strain is preserved in China center for type culture Collection, and the DF1 strain is preserved in a preservation No. CCTCC NO: knock-out of ornithine carbamoyl transferase gene OTC, L-ornithine-N5-transacetylase gene sidL, FC synthetase gene sidC and L-ornithine-N on the basis of Aureobasidium melanogenin HN6.2 strain of M2020340 ⁵ -the hydroxyl-transacetylase gene sidF.

Escherichia coli competence DH5 alpha, pMD19-T Simple vector purchased from Takara Bio Inc.; the map of the expression vector pAMEXloxp-1 is shown in figure 1, and the whole gene sequence is shown in SEQ ID NO. 7.

T4DNA ligase, restriction enzymes (SpeI, SalI and SmaI), DNA polymerase were purchased from Dalibao Bio Inc.

Plasmid DNA extraction kit and PCR purification kit were purchased from TSINGKE.

LB culture medium: 10g/L tryptone, 5g/L yeast extract powder and 10g/L sodium chloride, fully dissolving in deionized water, naturally adjusting the pH value, and adding 20g/L agar powder into a solid culture medium; autoclaving at 115 deg.C for 30 min; ampicillin was added at a final concentration of 100.0. mu.g/mL prior to use.

YPD medium: fully dissolving 20g/L peptone, 10g/L yeast extract and 20g/L glucose in deionized water, naturally adjusting the pH value, and adding 20g/L agar powder into a solid culture medium; autoclaving at 115 deg.C for 30min

Iron deficiency medium (MSP medium): 10-30g/L glucose, 1-5 g/L ammonium sulfate, 1-4 g/L dipotassium hydrogen phosphate, 1-2 g/L citric acid monohydrate, 1-5 g/L YNB, 0.01-0.1 g/L magnesium sulfate, 0.001-0.005 g/L zinc sulfate, pH 6-7, pH 6.5 adjustment, and autoclaving at 115 ℃ for 30 min. All glassware used in the preparation process needs to be soaked in 6.0N hydrochloric acid solution overnight, and then washed for several times by distilled water to remove iron attached to the glassware and avoid interference to experiments.

Piperazine acid production medium (CM medium): 10-50 g/L glucose, 10-30g/L peptone, 1-5 g/L dipotassium hydrogen phosphate, 1-2 g/L citric acid monohydrate, 1-5 g/L YNB, 0.01-0.1 g/L magnesium sulfate, 0.001-0.005 g/L zinc sulfate, and pH 6-7. Autoclaving at 115 deg.C for 30 min.

The invention is further illustrated by the following examples.

Example 1: transformation of piperazine synthase genes of different origins

Constructing a yeast strain expressing a piperazine synthase gene comprises:

six L-piperazine synthetase homologous genes from different sources obtained by NCBI are subjected to codon optimization and sent to a company for synthesis. Obtaining a series of recombinant plasmids after vector construction; the respective recombinant plasmids were transformed into DF1 strains, respectively, to obtain a series of strains expressing different sources of L-piperazine synthetase genes, as shown in table 1 below:

TABLE 1

1. Construction of recombinant plasmid:

(1) plasmids were synthesized by SalI and SpeI linearization;

(2) the linearized plasmids were ligated to the expression vector pAMEXloxp-1 by means of a recombinase, the specific procedure being as follows:

a. a connection system: pAMEXloxp-1 vector 2uL, gene fragment 15uL, Buffer 2uL, T4DNA Ligase 1 uL, and distilled water to make up to 20 uL. Mixing, reacting at 37 deg.C for 30min, and ice-cooling for 5 min.

b. The ligation products were transferred to 50. mu.L DH5a competent cells, incubated on ice for 20min, heat-shocked at 42 ℃ for 90S, added with 800. mu.L pre-cooled LB, incubated at 37 ℃ for 30min, then plated with 100ug/mL Amp resistant LB plates and incubated overnight in a 37 ℃ incubator.

c. Positive clones were picked up in LB tubes containing 100ug/mL Amp resistance and cultured overnight at 37 ℃ at 220 rpm.

d. And (3) extracting plasmids from the escherichia coli cells cultured in the test tube and sequencing. And comparing the sequencing results to obtain the correct recombinant plasmid.

And (3) transforming the series of recombinant plasmids with correct sequencing to be competent for yeast by a protoplast transformation method. And (3) coating the transformed yeast on a YPD solid culture medium containing a corresponding antibiotic for screening, culturing in an incubator at 28 ℃ for 2 days until a transformant appears, selecting a positive transformant, and performing subsequent fermentation after re-screening.

2. Transformation of recombinant plasmids

Protoplast transformation of a Strain

(1) A single colony of the Δ sidA strain activated for 48h on a YPD plate was inoculated into 50mL of HC liquid medium and cultured at 28 ℃ for 12h with shaking at 180 rpm.

(2) After microscopic examination to confirm no infectious microbes, 25mL of the bacterial solution containing more yeast-like bacteria is put into a sterile 50mL centrifuge tube, 8000 Xg is carried out, and the bacteria are centrifuged for 5min at room temperature and collected.

(3) The cells were resuspended in 30mL sorbitol-trisodium citrate buffer, 8000 Xg, centrifuged at room temperature for 5min, and collected.

(4) The cells were resuspended in 5mL sorbitol-trisodium citrate buffer, 1mL of digesting enzyme and 1mL of collapsing enzyme (supernatant was centrifuged before use) were added, and the cells were disrupted at 22 ℃ and 100rpm for 30-60 min.

(5) The treated cells were subjected to microscopic examination, and after confirming cell wall rupture, 3000 Xg was centrifuged for 5min to collect cells.

(6) The cells were resuspended in 30mL of STC buffer, 3000 Xg, centrifuged for 5min, and the supernatant was discarded.

(7) The cells obtained in the previous step were resuspended in 6mL of STC buffer, centrifuged at 3000rpm for 5min at room temperature, and the cells were collected.

(8) 1.5mL of STC buffer solution is taken to resuspend the thalli, and the competent cells are obtained.

(9) Add 50. mu.L of linearized fragment and 50. mu.L of PTC solution to 200. mu.L of competent cells in 1.5mL sterile centrifuge tube and mix well. Water bath at 22 deg.c for 10 min. Blank control group was not added with linearized fragments.

(10) Adding PTC buffer solution 50 μ L, 200 μ L, and 800 μ L into the mixture 3 times at 1min interval, and mixing gently.

(11) Keeping the temperature in a water bath at 22 ℃ for 1h, then centrifuging at 3000 Xg and 22 ℃ for 15min, and removing the supernatant.

(12) The cells were resuspended in 1mL of 2 XHCS buffer, 2000 Xg, and centrifuged at 22 ℃ for 15min to collect the cells.

(13) After resuspending the cells in 0.5mL of 2 XHCS buffer, the cells were cultured at 28 ℃ and 100rpm for 1 hour to double the cell walls. The cells after the wall covering are coated on a double-layer HCS solid culture medium (hygromycin B or nourseothricin with the concentration of 50 mu g/mL is added in the lower layer, and antibiotics are not added in the upper layer), and are statically cultured for 48 hours at 28 ℃.

(14) The transformants were picked and inoculated on YPD solid medium at 100. mu.g/mL hygromycin B or nourseothricin, and left to stand at 28 ℃ for 36 hours. And (4) preserving the transformant obtained by re-screening for the next screening and verification step.

Introducing each expression plasmid into DF1 by standard protoplast transformation method, fermenting by CM culture medium, carrying out Fmoc-CL derivatization on the fermentation product, and finally, observing whether a new peak corresponding to a standard product is generated by HPLC analysis, thereby preliminarily judging whether the transformed piperazine synthetase gene is the required target gene.

3. Fermentation product determination and screening of Yeast transformants

(1) The high performance liquid chromatography analysis method after fermentation product FDAA derivatization comprises the following steps:

flow rate: 0.5 mL/min;

wavelength: 340 nm;

column temperature: 30 degrees;

column: c18 water phase column;

the loading program settings are shown in table 2 below;

TABLE 2

(2) The high performance liquid chromatography analysis method after the fermentation product Fmoc-Cl derivatization comprises the following steps:

flow rate: 1 mL/min;

wavelength: 280 nm;

column temperature: 28 degrees;

column: phenomenex Luna C18 column (5 μm,4.6mm ID. times.250 mm);

the loading program settings are shown in table 3 below;

TABLE 3

(3) The highest yield of the piperazine synthetase gene KtzT is obtained by HPLC detection after the fermentation product Fmoc-CL derivatization (the result is shown in Table 4).

TABLE 4

(Note: Aureobasidium melanogenesis DOLCF, i.e., DF1 strain: a strain in which OTC, sidL, sidC and sidF were knocked out for the Aureobasidium melanogenesis original strain in order to accumulate more piperazinic acid precursors; strain DFK1-DFK 6: DF1 strain transformed with piperazinic acid synthase genes from different sources as shown in Table 1; DFAK1 strain DF1 was continuously knocked out for relieving iron ion inhibition ⁵ Hydroxylase gene sidA, followed by co-expression of sidA provided according to the invention and piperazine acid synthesis gene ktzT, resulting in strain DFAK 1. )

As is apparent from Table 3, the highest yield of crude piperazinic acid was 63.61mg/L for the strains derived from the L-piperazinic acid synthetase genes, which is far less than 152.98mg/L and 179.37mg/L for the strains of the present invention (DFK1 and DFK2), and the microbial strains DFK1 and DFK2 of the present invention have a very significant advantage in yield as compared with yeast strains derived from the L-piperazinic acid synthetase genes, which also indicates that the L-piperazinic acid synthetase genes derived from different sources are greatly different from each other in yield.

(4) Separation and purification and nuclear magnetic identification

The derivatized crude product was further purified by semi-preparative liquid phase according to HPLC procedure after derivatization of the conversion product Fmoc-Cl as in example 1, wherein the liquid feed was 200uL, using an elette C18 reverse phase preparative column. Manually intercepting a peak at a target position, connecting an EP tube every 1mL after the target peak appears, then detecting by HPLC analysis, and concentrating by a rotary evaporator to remove derivatization to obtain 11.25mg of brown solid.

The obtained solid is treated with D ₂ Dissolving O, moving to a sample bottle, and carrying out nuclear magnetic detection. The detection items include one dimension ( ¹ H、 ¹³ C) And two-dimensional (COSY (H-H), HMBC, HSQC). The L-piperazinoic acid is determined by mass spectrometry, nuclear magnetic resonance and liquid phase absolute typing (see the description in FIGS. 2 to 7).

Example 2: construction of SidA-KtzT Co-expression Strain

Constructing a recombinant plasmid pAMEXloxp-1-SidA-KtzT containing an L-ornithine hydroxylase coding gene SidA and an L-piperazine synthetase coding gene ktzT according to the method;

the constructed recombinant plasmid pAMEXloxp-1-SidA-KtzT is transformed into DF1 host bacteria of which the original SidA is knocked out by an electrotransformation method, and the DF1 host bacteria are coated on a YPD solid medium plate containing corresponding antibiotics and cultured overnight at 28 ℃ to construct a genetic engineering bacterium DFAK 1.

DFAK1 was fermented in CM medium for 5 days, and the piperazine acid yield was 694.87. + -. 41.36mg/L by HPLC, and the results are shown in Table 3.

Example 3: effect of iron ion on piperazine acid production

In order to verify whether the strain DFAK1 is relieved of iron ion inhibition, in the experiment, the yields of piperazine acid were measured after fermentation of DFK1 and DFAK1 in MSP medium and high-iron medium (adding ferric chloride with a final concentration of 10 μ M based on MSP) for 5d and 120h, respectively, and the results are shown in FIG. 8, from which it can be seen that the strain DFAK1 is completely relieved of iron ion inhibition.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

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ttcgataagg tcaccggcac tgtctctaac accggtaaca cttggttcaa gcttctgatc 540

aagcctggct gcgactctac tgaggaagag ggcgatgctt ggtacttgcg cccaggcgat 600

gtcgtccacc agcccgagct ccgtcaacct ggtaaccact accttgtcta caacgacaag 660

ttcatcaaga tctctgattc ttgcccagct aagcctccat ctgccgatta a 711

<210> 6

<211> 678

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atggtcgtcg gtatgaccgc tgagacctct actgtccagg gcgctctttt cggtgaccct 60

gccgtcatcg agaccactga tcacggccct accgccactc aggacccacg tgaggtcgct 120

agagtcgtcg gccttgccca agatccaggt ctgttcgtcg tcgagagaac tggtcttgtc 180

ctgcgcgctg atccagctag acctggttgc gctgatgctc tggctcgtca cgacggtgat 240

accgtcgctc aactccttga tactggccac ctcagacttg gcggtaccca ccatgtccat 300

cacaacggtg atgagggtcc agctcgttct gtcctcgtcc ctaaggccac tagagacatg 360

gtctctcgct gggatcacct tcgtcctatc ccagagccca ccagagctcc tgagccaact 420

aaggccccac agagatctac tggtctcatc ggtgtcgatg tcgtcgagcc tggcaaggct 480

ctggtctgct tgggtaccac tggtcaaggc ggtactgtcc tgcgcgatgg cggtagatac 540

cgcgtcgaga acgaccatgg tgctttggtc ggtcatgctt cttcttaccg cgctgccgct 600

cgtctgctcg ctagatacca cggctacgcc cccggtcctg tcgagatcga gcacgagcac 660

cgtcttcacc gccgttaa 678

<210> 7

<211> 5424

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 60

cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 120

tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 180

aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 240

ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 300

ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 360

tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 420

tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 480

actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 540

gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 600

acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 660

gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 720

acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 780

gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 840

ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 900

gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 960

cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 1020

agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 1080

catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 1140

tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 1200

cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 1260

gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 1320

taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 1380

ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 1440

tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 1500

ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 1560

cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 1620

agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 1680

gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 1740

atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 1800

gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 1860

gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 1920

ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 1980

cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 2040

cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 2100

acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 2160

cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 2220

accatgatta cgccaagctt gcatgccccg gggctaaaaa ccccaacttc ggaaaggggt 2280

gtatttatta gataaaaaac caacgccttt cggggctcct tggtgattca taataactaa 2340

acgaatcgca tggccttgcg ccggcgatgg ttcattcaaa tttctgccct atcaactttc 2400

gatggtagga tagtggccta ccatggtatc aacgggtaac ggggaattag ggttctattc 2460

cggagaggga gcctgagaaa cggctaccac atccaaggaa ggcagcaggc gcgcaaatta 2520

cccaatcccg acacggggag gtagtgacaa taaatactga tacagggctc ttttgggtct 2580

tgtaattgga atgagtacaa tttaaatccc ttaacgagga acaattggag ggcaagtctg 2640

gtgccagcag ccgcggtaat tccagctcca atagcgtata ttaaagttgt tgcagttaaa 2700

aagctcgtag ttgaaccgac agttggctca tcatccgtta catcatcttt ttttctgctg 2760

gctccgctcg gtgggctccc aacgaagcag caaaaaaagt gagagagaaa aactagcttg 2820

gcggggcatc cgaagctaga ccctttggct cgcttagtca gtgcgccctc tcacacacac 2880

tcaaaaaggc cacccctccc gcaccctctt ctcatcaccg tcttcatacc acggttcgtc 2940

aagcaatcgt atctggtaag ctttgacctc ttgagcgggc tccactttgc tatttcttgg 3000

atctgctcat tcttttcttc cttcctctct ttctaacctc tcttcagaaa gttcaaccgt 3060

acttcactca atcttccata catcaccgtc aaacactagt cttaagatgc atacgcgtga 3120

gctcctgcag ggtaccgtcg actaccgttc gtatagcata cattatacga agttattaat 3180

tcgggggatc tggattttag tactggattt tggttttagg aattagaaat tttattgata 3240

gaagtatttt acaaatacaa atacatacta agggtttctt atatgctcaa cacatgagcg 3300

aaaccctata ggaaccctaa ttcccttatc tgggaactac tcacacatta ttatggagaa 3360

actcgagctt gtcgatcgac agatcccggt cggcatctac tttatggaca tggcatggac 3420

atgtacaaag cctgttcacc atcagaagca gtaccgtcgt acaaagcagt atccaaacca 3480

cacaaggtga aacccattct tctgtaagcg tggatagctg gagcgttgac gttggtaacc 3540

tccaaccaca agtgaccagc acctctctct ctagcgaact cagtagccaa acccatcaaa 3600

gctctaccga caccgtgacc tctgtgttct ggagcgacct cgatgtcttc aacggtcaat 3660

cttctgttcc aaccggagta agagacaacg acgaaaccag ccaaatcacc atcgtcaccg 3720

taagcgacga aagttctgga gtctggatca ccgtcctcac cagcgtcgga ctcatcgtca 3780

gattcatcgt ctgggaaaac cttggtcaat ggtggatcga ctggaacttc tctcaaagtg 3840

aaaccgtcac cagtagcggt gactctgaaa acagtatcag tggtgaagga accgtccaaa 3900

gcctcgatag cttcagcatc acctggaaca gaagttctgt atctgtaagc ggtatcgtcc 3960

aaagtggtca ttgtgactga attggatgtg ttagacaggt tgatgtgaac gacagcaaca 4020

gagaaagacg cacagagtta tatacaacag agcttgatgg gttgttgtgg tgtgtgaggg 4080

gcttgaatct ttccccaaca gcttccgacg ctcggcatga ggggcagctc aaaaaaaccg 4140

cgtcaccgac gtgctaccga gatcttcgtg ctctctcgtg tttgcctatc aactgatatc 4200

accaatcatg agccatctct atgtacaaac tgcccttgga tcacgtgctt cactcgacat 4260

ctgcaccatg tatccttctt cgaattgctc atcttgatca ctttattatt caagatgcct 4320

tgcaccacac gacatgtcta tcgatcggcc cgtaacgccc cgtgaagttg ccagaacgcc 4380

tgcctgtccc gctgtccgat ggatgcatga aggtaatata acttcgtata gcatacatta 4440

tacgaacggt aggatcccca acagggattg ccctagtaac ggcgagtgaa gcggcaacag 4500

ctcaaatttg aaagctggcc ttcgggtccg cattgtaatt tgtagaggat gctttgggtg 4560

aaacgccagt ctaagttcct tggaacagga cgtcatagag ggtgagaatc ccgtatgtga 4620

ctggaaatgt taacctatgt aaagctcctt cgacgagtcg agttgtttgg gaatgcagct 4680

ctaaatggga ggtaaatttc ttctaaagct aaatattggc gagagaccga tagcgcacaa 4740

gtagagtgat cgaaagatga aaagcacttt ggtaagagag ttaaaaagca cgtgaaattg 4800

ttgaaaggga agcgcttgca atcagacttg tttaaactgt tcggccggtc ttctgaccgg 4860

tttactcagt ttggacaggc cagcatcagt ttcggcggcc ggataaaggc tttgggaatg 4920

tggccttcac ttcggtgaag gtgttgtagc ccagggtgta atacggccag ccgggactga 4980

ggtccgcgct tcggctagga tgctggcgta atggttggaa ttccccggga ctggccgtcg 5040

ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac 5100

atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac 5160

agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt 5220

gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt 5280

taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc 5340

cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt 5400

caccgtcatc accgaaacgc gcga 5424

<210> 8

<211> 1461

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atgtcccgac tgacacaatc caaagaagaa agaaagataa tcaacacaat ggcacctcac 60

gccgacattt ccgaaattgc tctcgagagc gtcccagagc gcaatggctc cagattcaaa 120

acacacctcc agtcaacacc acagaacgac gtgcacgatg tagtctgcat tggcttcggt 180

tctgcctcgc tggcaatcgc agttgccctc cacgatgcga ccaagcagac gggcaagcag 240

ccaccacaac tcagaacaca acccaaggtc gccttcctcg agaagcaaga gacgttcgca 300

tggcacgctg gcatgcagat tcctggtgcc aagatgcaaa tctcgttcat caaggacatg 360

gccaccatgc gcaaccctcg cagcgagttc accttcctca actacctgca cgagaacgac 420

cgcctggtgc agttcaccaa cctcagcact ttcctgcctc ggcgcattga gttcgaggac 480

tatctccgct ggtgtgccgg ctggttccag aacctggttt catatggtca agaggtggtc 540

gatatctcgc ctgacacgca ggccaagagt gcagagggtg tgacaacgtg gacaatcaca 600

tctcgcaacc ttcacaccaa cgaactcagc ttccgccgca cgaaacgcgt tgtcgttgct 660

gtcggaggtc acccaagaat cccagaggcc ttccctcaac accacccccg tgtcctccac 720

tcgtcgcaat actggactct ctcgtcccgc atcttccgcg accgtaatgc tcctctcaaa 780

atcgccgtca ttggctctgg tcaatccgct gccgagatct tcatgaactt gcccagccaa 840

ttccccaact ccaaagccta cctcatgatc agaggcgctg ccctcagacc ttcggatgac 900

tcgcctttcg tcaacgaagt cttcgacccc gagcgtacta atgacacctt cagtcaagat 960

cctgagatcc gtgccgcagc catcaagatg gacaaggcca ccaactacgg tgtcgttcgt 1020

ctcgagctgc tggagcacat gtatgatgtc ctctatacac aacgcctcaa ccacggcgac 1080

gatgaggaga actggcccca acgcctgctc aactaccgcg aagtcgagag tgtgaccgaa 1140

ttgcccgacg accgtgtccg actccacatc agaaacgata cctcgaagca cagatgtcgc 1200

aagagctcta cgaaagagac tctggatgtg gatctggtca tgatcgccag cggatacaga 1260

agagatcacc acgagcagtt gttgtccaag gtcagaccca tgatgcccgg tggggacaag 1320

cctggtcaga agtggaccgt caaccgcgaa tacaaggtcg agtttgagca gggcactgtc 1380

gcaaaggact ctggtgtctg gttgcaggga tgtaacgaga gtacccacgg tgtaagtttt 1440

ggtccaacct tcgcttcgtg a 1461

Claims (8)

1. A microbial strain for efficiently synthesizing L-piperazinic acid is characterized in that a carrier containing an L-piperazinic acid synthetase encoding gene ktzT or hmtC is further transformed in a aureobasidium melanoidinum D-OLCF strain, or the L-piperazinic acid synthetase encoding gene ktzT or hmtC is integrated into a genome of the aureobasidium melanoidinum D-OLCF strain;

the coding gene ktzT of the L-piperazine synthetase is derived from Kutzneria sp.744;

the gene hmtc encoding the L-piperazine synthetase is derived from Streptomyces himastatinicus ATCC 53653;

the aureobasidium pullulans D-OLCF is a compound with the preservation number of CCTCC NO: knock-out of ornithine carbamoyl transferase gene OTC and L-ornithine-N on the basis of Aureobasidium melanogenin HN6.2 strain of M2020340 ⁵ Transacetylase gene sidL, FC synthetase genesindC and L-ornithine-N ⁵ -a strain of the hydroxy-transacetylase gene sidF.

2. The microorganism strain according to claim 1, further comprising a vector comprising a gene sidA encoding L-ornithine hydroxylase shown in SEQ ID NO. 8, which is obtained by knocking out the gene sidA encoding the original L-ornithine hydroxylase in the aureobasidium pullulans D-OLCF producing strain; or replacing the original gene sidA encoding the L-ornithine hydroxylase by the gene SidA encoding the L-ornithine hydroxylase shown in SEQ ID NO. 8 in the genome of aureobasidium pullulans D-OLCF.

3. Use of the microorganism strain according to claim 1 or 2 for the fermentative production of L-piperazinoic acid or for the preparation of a microbial product for the fermentative production of L-piperazinoic acid.

4. The method for constructing a microorganism strain according to claim 1, wherein the microorganism strain is obtained by constructing a vector containing the gene ktzT or hmtc encoding L-piperazinic acid synthetase and then transforming the vector into a strain producing aureobasidium melanophore D-OLCF.

5. The construction method according to claim 4, wherein the vector is pAMEXloxp-1 plasmid, and the whole gene sequence is shown in SEQ ID NO. 7.

6. The construction method according to claim 4 or 5, further comprising knocking out the gene sidA encoding L-ornithine hydroxylase present in the aureobasidium melanophore D-OLCF strain and converting the vector containing the gene sidA encoding L-ornithine hydroxylase as shown in SEQ ID NO. 8; or replacing the original L-ornithine hydroxylase coding gene sidA with the L-ornithine hydroxylase coding gene SidA shown in SEQ ID NO. 8 in the aureobasidium pullulans D-OLCF genome.

7. A method for producing L-piperazinic acid by fermentation, comprising inoculating the microorganism strain of claim 1 or 2 to a piperazinic acid-producing medium to produce L-piperazinic acid by fermentation.

8. The method of claim 7, wherein the piperazinoic acid producing medium is CM medium.

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