阅读说明：本技术 分离的多肽及其应用 (Isolated polypeptides and uses thereof ) 是由 刘然 于 2018-06-25 设计创作，主要内容包括：本发明公开了分离的多肽及其应用。其中,该分离的多肽包含nisB序列和前体肽序列,其中,所述nisB序列与SEQ ID NO：2所示的氨基酸序列具有至少90％一致性,且部分序列高度保守,且具有催化所述前体肽序列形成的肽段脱水的活性；所述前体肽序列与SEQ ID NO：3所示的氨基酸序列具有至少90％一致性,且部分序列高度保守,且具有与所述nisB序列形成的肽段结合并被脱水修饰的活性。通过表达nisin的产量依赖敏感的NisB序列和前体肽序列,使nisin的产量显著提高。(The invention discloses an isolated polypeptide and application thereof. Wherein the isolated polypeptide comprises a nisB sequence and a precursor peptide sequence, wherein the nisB sequence is identical to the sequence of SEQ ID NO:2, the amino acid sequence has at least 90 percent of consistency, part of the sequence is highly conserved, and the amino acid sequence has the activity of catalyzing the dehydration of the peptide segment formed by the precursor peptide sequence; the precursor peptide sequence is identical to SEQ ID NO: 3, and a part of the sequence is highly conserved and has the activity of being combined with a peptide fragment formed by the nisB sequence and being dehydrated and modified. The yield of nisin is obviously improved by expressing that the yield of nisin depends on sensitive NisB sequences and precursor peptide sequences.)

1. An isolated polypeptide comprising a nisB sequence and a precursor peptide sequence, wherein,

the nisB sequence is similar to SEQ ID NO:2, and has at least 90% identity to the amino acid sequence set forth in SEQ ID NO:2, arginine at position 83, arginine at position 87, threonine at position 89, aspartic acid at position 121, arginine at position 154, isoleucine at position 171, valine at position 176, valine at position 198, tyrosine at position 202, leucine at position 209, tyrosine at position 213, leucine at position 217, aspartic acid at position 299, arginine at position 464, arginine at position 786, arginine at position 826 and histidine at position 961, and has the activity of catalyzing the dehydration of a peptide fragment formed by the precursor peptide sequence;

the precursor peptide sequence is identical to SEQ ID NO: 3 and has at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 3, the amino acid sequence has the activity of combining with a peptide fragment formed by the nisB sequence and being modified by dehydration, wherein the 6 th to the 9 th positions of the amino acid sequence are phenylalanine-asparagine-leucine-aspartic acid, the 26 th position is serine, the 28 th to the 34 th positions are serine-leucine-cysteine-threonine-proline-glycine-cysteine, the 36 th position is threonine, the 37 th position is glycine, the 39 th position is leucine, the 42 th position is cysteine, the 46 th position is threonine, the 48 th position is threonine, the 49 th position is cysteine, and the 51 st position is cysteine.

2. An isolated nucleic acid encoding the polypeptide of claim 1.

3. The nucleic acid of claim 2, wherein the nucleic acid further comprises:

a Pnis promoter gene having the nucleotide sequence of SEQ ID NO: 1.

4. A recombinant vector comprising the nucleic acid of claim 2 or 3.

5. The recombinant vector according to claim 4, wherein the recombinant vector is a plasmid.

6. A recombinant cell comprising the recombinant vector of claim 4 or 5.

7. The recombinant cell of claim 6, wherein the recombinant cell is a lactic acid bacterium.

8. A method for the preparation of nisin, characterized in that engineered cells overexpress the nisB sequence or co-express the nisB sequence and the precursor peptide sequence.

9. The method of claim 8, wherein the engineered cell is the recombinant cell of claim 6 or 7.

10. The method of claim 9, wherein the engineered cell is a lactic acid bacterium comprising the recombinant vector of claim 4 or 5.

Technical Field

The present invention relates to the field of bioengineering, in particular to isolated polypeptides and uses thereof, more particularly to isolated polypeptides, nucleic acids, recombinant vectors, recombinant cells and methods for producing nisin.

Background

Nisin (Nisin) is a natural active bacteriostatic polypeptide produced by lactic acid bacteria, and belongs to the family of lantibiotics. Mature Nisin is composed of 34 amino acids, has a strong inhibitory effect on most gram-positive bacteria and spores thereof, has a killing effect on gram-negative bacteria when acting together with EDTA and the like, and is the only bacteriocin approved for food preservation. Nisin is particularly sensitive to protease, can be quickly decomposed by alpha-chymotrypsin in the digestive tract without influencing normal flora in the intestinal tract, is basically non-toxic to human bodies, and does not generate cross drug resistance with medical antibiotics. The compound has become a green food additive with great prospect and a potential antibiotic substitute, and is widely applied to the fields of food industry and biomedicine. Nisin can be obtained by lactic acid bacteria fermentation at present, but the yield is low, and the production requirement cannot be met.

Thus, a method for preparing nisin in high yield is yet to be studied.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art. The inventor finds out that the key speed-limiting step of nisin synthesis is the NisB dehydration step through an in-vitro reconstruction system, and remarkably improves the output of nisin generated by fermentation by over-expressing the nisin synthesis substrate precursor peptide and nisB.

It should be noted that the present invention is completed based on the following work of the inventors:

nisin is biosynthesized in a pathway shown in FIG. 1, the precursor peptide nisZ or nisA is dehydrated by NisB, NisC is cyclized to generate a lanthionine ring, and the leader peptide is removed by NisP to finally form mature Nisin.

The inventors investigated the effect of each step in the pathway for nisin biosynthesis shown in FIG. 1 on nisin production by in vitro reconstitution. It was found that, under the conditions of limiting the energy supplied in the system, reducing power, etc., nisin production increased significantly with increasing concentration of the precursor peptide and stabilized between 0.4 and 1.3nM, slowly with increasing concentration of the precursor peptide, and nisin production was high with limiting the concentration of the precursor peptide between 0.4 and 1.3 nM. While the NisP-encoding plasmid concentration had no significant effect on nisin production. However, when the concentration of NisB was varied from 10 to 1500nM, the production of active nisin Z increased significantly with NisB concentration. Whereas the production of active Nisin Z is sensitive to NisC content in a rather low concentration range of 1 to 100 nM. More than a 10-fold change in nisin production was observed between 1nM and 100nM NisC, whereas higher concentrations of NisC (from 100 to 10000nM) did not significantly affect nisin production. Therefore, in the process of preparing nisin by fermentation, the concentrations of NisB and the precursor peptide directly influence the yield of nisin, the inventor improves the yield of nisin of lactic acid bacteria by enabling lactic acid bacteria to over-express NisB and the precursor peptide, and experiments show that the yield of lactic acid bacteria over-expressing NisB and the precursor peptide is improved by 60 percent relative to the original yield of lactic acid bacteria.

Thus, according to a first aspect of the invention, there is provided an isolated polypeptide. According to an embodiment of the invention, the isolated polypeptide comprises a nisB sequence and a pre-peptide sequence, wherein,

the nisB sequence is similar to SEQ ID NO:2, and has at least 90% identity to the amino acid sequence set forth in SEQ ID NO:2, arginine at position 83, arginine at position 87, threonine at position 89, aspartic acid at position 121, arginine at position 154, isoleucine at position 171, valine at position 176, valine at position 198, tyrosine at position 202, leucine at position 209, tyrosine at position 213, leucine at position 217, aspartic acid at position 299, arginine at position 464, arginine at position 786, arginine at position 826 and histidine at position 961, and has the activity of catalyzing the dehydration of a peptide fragment formed by the precursor peptide sequence;

the precursor peptide sequence is identical to SEQ ID NO: 3 and has at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 3, wherein the amino acid sequence is phenylalanine-asparagine-leucine-aspartic acid at positions 6 to 9, serine at position 26, serine-leucine-cysteine-threonine-proline-glycine-cysteine at positions 28 to 34, threonine at position 36, glycine at position 37, leucine at position 39, cysteine at position 42, threonine at position 46, threonine at position 48, cysteine at position 49, cysteine at position 51, and has an activity of binding to a peptide fragment formed by the nisB sequence and being modified by dehydration, wherein SEQ ID NO:2 and SEQ ID NO: 3 is as follows:

MIKSSFKAQPFLVRNTILSPNDKRSFTEYTQVIETVSKNKVFLEQLLLANPKLYNVMQKYNAGLLKKKRVKKLFESIYKYYKRSYLRSTPFGLFSETSIGVFSKSSQYKLMGKTTKGIRLDTQWLIRLVHKMEVDFSKKLSFTRNNANYKFGDRVFQVYTINSSELEEVNIKYTNVYQIISEFCENDYQKYEDICETVTLCYGDEYRELSEQYLGSLIVNHYLISNLQKDLLSDFSWDTFLTKVEAIDEDKKYIIPLKKVQKFIQEYSEIEIGEGIEKLKEIYQEMSQILENDNYIQIDLISDSEINFDVKQKQQLEHLAEFLGNTTKSVRRTYLDDYKDKFIEKYGVDQEVQITELFDSTFGIGAPYNYNHPRNDFYESEPSTLYYSEEEREKYLSMYVEAVKNHNVINLDDLESHYQKMDLEKKSELQGLELFLNLAKEYEKDIFILGDIVGNNNLGGASGRFSALSPELTSYHRTIVDSVERENENKEITSCEIVFLPENIRHANVMHTSIMRRKVLPFFTSTSHNEVQLTNIYIGIDEKEKFYARDISTQEVLKFYITSMYNKTLFSNELRFLYEISLDDKFGNLPWELIYRDFDYIPRLVFDEIVISPAKWKIWGRDVNNKMTIRELIQSKEIPKEFYIVNGDNKVYLSQENPLDMEILESAIKKSSKRKDFIELQEYFEDENIINKGQKGRVADVVVPFIRTRALGNEGRAFIREKRVSVERREKLPFNEWLYLKLYISINRQNEFLLSYLPDIQKIVANLGGKLFFLRYTDPKPHIRLRIKCSDLFLAYGSILEILKRSQKNRIMSTFDISIYDQEVERYGGFDTLELSEAIFCADSKIIPNLLTLIKDTNNDWKVDDVSILVNYLYLKCFFQNDNKKILNFLNLVSPKKVKENVNEKIEHYLKLLKVDNLGDQIFYDKNFKELKHAIKNLFLKMIAQDFELQKVYSIIDSIIHVHNNRLIGIERDKEKLIYYTLQRLFVSEEYMK(SEQ IDNO：2)

MSTKDFNLDLVSVSKKDSGASPRITSISLCTPGCKTGALMGCNMKTATCNCSIHVSK(SEQ ID NO：3)

according to the isolated polypeptide of the embodiment of the invention, the yield of nisin is obviously improved by expressing that the yield of nisin depends on sensitive nisib and precursor peptide.

According to a second aspect of the invention, there is provided an isolated nucleic acid. According to an embodiment of the invention, the nucleic acid encodes a polypeptide as described above. The nucleic acid has all the technical features and technical effects of the aforementioned polypeptide, which are not described herein again.

According to an embodiment of the invention, the nucleic acid further comprises: a Pnis promoter sequence having the sequence of SEQ ID NO:1, wherein the nucleotide sequence of the Pnis promoter sequence is specifically as follows:

TAATATCTTGATTTTCTAGTTCCTGAATAATATAGATATAGGTTTATTGAGTCTTAGACATAATTGAATGACCTAGTCTTATAACTATACTGACAATAGAAACATTAACAAATCTAAAACAGTCTTAATTCTATCTTGAGAAAGTATTGGCAATAATATTATTGTCGATAACGCGATCATAATAAACGGCTCTGATTAAATTCTGAAGTTTGTTAGATACAATGATTTCGTTCGAAGGAACTACAAAATAAATTATAAGGAGGCACTCAAA(SEQ ID NO：1)

according to a third aspect of the present invention, there is provided a recombinant vector. According to an embodiment of the invention, the recombinant vector contains the aforementioned nucleic acid. The vector can be obtained, for example, by inserting the above-mentioned nucleotide sequence into a cloning vector or an expression vector, or can be obtained by artificial synthesis. For example, the vector may be a plasmid.

According to a fourth aspect of the invention, there is provided a recombinant cell. According to an embodiment of the present invention, the recombinant cell contains the aforementioned recombinant vector. The recombinant cell has all the technical features and technical effects of the nucleic acid described above, and will not be described herein.

According to an embodiment of the present invention, the recombinant cell can be obtained by transforming the aforementioned vector into a host cell.

According to an embodiment of the invention, the recombinant cell is a lactic acid bacterium.

According to a fifth aspect of the present invention, there is provided a method for preparing nisin. According to embodiments of the invention, the engineered cell overexpresses the nisB sequence or coexpresses the nisB sequence and the precursor peptide sequence. Since the concentrations of nisB and the precursor peptide directly affect the yield of nisin during the preparation of nisin by fermentation, the inventors have increased the yield of nisin by overexpressing nisB and the precursor peptide in the engineered cells.

According to an embodiment of the invention, the engineered cell is a recombinant cell as described above. According to a preferred embodiment of the invention, the engineered cell is a lactic acid bacterium containing the recombinant vector described above.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 shows a schematic representation of the biosynthetic pathway of Nisin;

FIG. 2 shows a schematic representation of the amino acid sequences of various precursor peptides;

FIG. 3 shows a schematic ion flow chromatogram of nisin Z according to one embodiment of the invention;

FIG. 4 shows a schematic representation of the production of nisin Z according to one embodiment of the invention;

FIG. 5 shows a schematic representation of the production of nisin according to one embodiment of the invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.

It should be noted that the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Further, in the description of the present invention, "a plurality" means two or more unless otherwise specified.

Isolated polypeptide, nucleic acid, recombinant vector, recombinant cell

According to a first aspect of the invention, the invention provides an isolated polypeptide. In accordance with an embodiment of the present invention,

the isolated polypeptide comprises a nisB sequence and a precursor peptide sequence, wherein the nisB sequence is identical to SEQ ID NO:2, and has at least 90% identity to the amino acid sequence set forth in SEQ ID NO:2, arginine at position 83, arginine at position 87, threonine at position 89, aspartic acid at position 121, arginine at position 154, isoleucine at position 171, valine at position 176, valine at position 198, tyrosine at position 202, leucine at position 209, tyrosine at position 213, leucine at position 217, aspartic acid at position 299, arginine at position 464, arginine at position 786, arginine at position 826 and histidine at position 961, and has the activity of catalyzing the dehydration of a peptide fragment formed by the precursor peptide gene; the precursor peptide sequence is identical to SEQ ID NO: 3 and has at least 90% identity to the amino acid sequence set forth in SEQ id no: 3, the amino acid sequence has the activity of combining with a peptide fragment formed by the nisB sequence and being modified by dehydration, wherein the 6 th to the 9 th positions of the amino acid sequence are phenylalanine-asparagine-leucine-aspartic acid, the 26 th position is serine, the 28 th to the 34 th positions are serine-leucine-cysteine-threonine-proline-glycine-cysteine, the 36 th position is threonine, the 37 th position is glycine, the 39 th position is leucine, the 42 th position is cysteine, the 46 th position is threonine, the 48 th position is threonine, the 49 th position is cysteine, and the 51 st position is cysteine.

Wherein, it is to be noted that, the person skilled in the art can compare the nucleotide sequence of SEQ ID NO:2 and the amino acid sequence shown in SEQ ID NO: 3, but the modified sequence has at least 90% identity to the original amino acid sequence, e.g., may be 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

Specifically, the sequences of the precursor peptides are illustrated, and the precursor peptides are multiple, but have similar sequences, wherein the sequences shown in SEQ ID NOs: 3 is the amino acid sequence of the nisZ polypeptide, and the amino acid at the 50 th position is replaced, so that asparagine (N) is replaced by histidine (H), and another leader peptide nisA is obtained, wherein the specific sequence is as follows:

MSTKDFNLDLVSVSKKDSGASPRITSISLCTPGCKTGALMGCNMKTATCHCSIHVSK(SEQ ID NO：4)

leader nisA to SEQ ID NO: 3, or can be combined with nisB and modified and dehydrated under the catalysis of nisB.

Also, other leader peptides can be obtained by substituting or deleting other sites of the leader peptide, as shown in detail in FIG. 2, but it is to be noted that various modified leader peptides, some of the key active sites are not changed, that is, the leader peptide of SEQ ID NO:2, wherein the amino acid sequence is arginine at position 83, arginine at position 87, threonine at position 89, aspartic acid at position 121, arginine at position 154, isoleucine at position 171, valine at position 176, valine at position 198, tyrosine at position 202, leucine at position 209, tyrosine at position 213, leucine at position 217, aspartic acid at position 299, arginine at position 464, arginine at position 786, arginine at position 826 and histidine at position 961, that is, the amino acids are always maintained when individual amino acids in the amino acid sequence are modified, substituted, added and deleted.

According to the separated polypeptide disclosed by the embodiment of the invention, the yield of nisin is obviously improved by expressing that the yield of nisin depends on sensitive nisiB and precursor peptide; meanwhile, the Pnis promoter sequence is used as the promoter of the nisB and the precursor peptide, and further provides expression of the nisB and the precursor peptide, so that the yield of nisin is correspondingly further improved. According to an embodiment of the present invention, the yield of lactic acid bacteria overexpressing nisB and the precursor peptide is improved by up to 60% compared to the original lactic acid bacteria.

According to an embodiment of the invention, the polypeptide expression of the nisB protein and the precursor peptide is carried out using the Pnis promoter as promoter.

According to a second aspect of the invention, there is provided an isolated nucleic acid. According to an embodiment of the invention, the nucleic acid encodes a polypeptide as described above. The nucleic acid has all the technical features and technical effects of the aforementioned polypeptide, which are not described herein again.

The term "nucleic acid" used in the present invention may be any polymer containing deoxyribonucleotides or ribonucleotides, including but not limited to modified or unmodified DNA, RNA, the length of which is not subject to any particular limitation.

According to an embodiment of the invention, the nucleic acid further comprises: a Pnis promoter gene having the nucleotide sequence of SEQ ID NO:1, and optionally a Pnis promoter gene, a nisB gene (a gene encoding the nisB sequence) and a propeptide gene (a gene encoding the propeptide sequence). That is, the nisB gene and the precursor peptide gene are expressed using the Pnis promoter as a promoter, so that the expression level of the nisB gene and the precursor peptide gene is up-regulated, and the nisin yield is higher.

Wherein, the Pnis promoter gene, the NisB gene and the precursor peptide gene can be directly connected, namely, the 3 'end of the SEQ ID NO. 1 sequence is directly connected with the 5' end of the SEQ ID NO. 2 sequence, and so on; it may also be indirectly linked, for example, there may be other nucleotide sequences between the Pnis promoter gene and the NisB gene, as long as the expression of the NisB gene and the propeptide gene is not affected. Furthermore, the Pnis promoter gene, NisB gene and propeptide gene may be linked in any order, i.e., they may be Pnis promoter gene-NisB gene-propeptide gene, or Pnis promoter gene-propeptide gene-NisB gene.

According to a third aspect of the present invention, there is provided a recombinant vector. According to an embodiment of the invention, the recombinant vector contains the aforementioned nucleic acid.

The term "recombinant vector" as used in the present invention refers to a genetic vector comprising a specific nucleic acid sequence and capable of transferring a nucleic acid sequence of interest into a host cell to obtain a recombinant cell. According to an embodiment of the present invention, the form of the recombinant vector is not particularly limited.

According to an embodiment of the present invention, the recombinant vector may be a plasmid. The plasmid is used as a genetic carrier, has the characteristics of simple operation, capability of carrying larger fragments and convenience for operation and treatment. The form of the plasmid is not particularly limited, and may be a circular plasmid or a linear plasmid, and may be either single-stranded or double-stranded. The virus is easily transfected into recipient cells. The skilled person can select as desired. For recombinant vectors used to construct recombinant cells, it is preferred that the nucleic acid be DNA, as DNA is more stable and easier to manipulate than RNA.

According to a fourth aspect of the invention, there is provided a recombinant cell. According to an embodiment of the present invention, the recombinant cell contains the aforementioned recombinant vector.

According to an embodiment of the present invention, the recombinant cell can be obtained by transforming the aforementioned vector into a host cell.

At present, nisin is mainly prepared by lactic acid bacteria fermentation, and has high yield and low cost. Accordingly, according to an embodiment of the invention, the recombinant cell is a lactic acid bacterium. Therefore, the lactic acid bacteria are used as recombinant cells, the activity of the cells is high, the yield of nisin is high, and the cost is low.

Process for preparing nisin

According to a fifth aspect of the present invention, there is provided a method for preparing nisin. According to embodiments of the invention, the engineered cell overexpresses the nisB sequence or coexpresses the nisB sequence and the precursor peptide sequence. Since the concentrations of NisB and the precursor peptide directly affect the yield of nisin during the nisin preparation process by fermentation, the inventors have improved the yield of nisin by allowing engineered cells to overexpress NisB and the precursor peptide.

According to an embodiment of the invention, the engineered cell is a recombinant cell as described above. The recombinant cell contains nisB gene and precursor peptide gene, the promoter is Pnis promoter, the expression quantity of nisB gene and precursor peptide gene is up-regulated, and the improvement of the yield of nisin is facilitated. Since existing nisin is generally obtained by fermentation using lactic acid bacteria, it is preferable to use lactic acid bacteria as recombinant cells, that is, lactic acid bacteria containing the aforementioned recombinant vector. Thus, the yield of nisin was higher.

The present invention is described below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention.

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are carried out according to techniques or conditions described in literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruke et al, Huang Petang et al) or according to product instructions. The reagents or apparatus used are conventional products which are commercially available, e.g. from Sigma, without reference to the manufacturer.