CN117586356B

CN117586356B - Polypeptides and uses thereof

Info

Publication number: CN117586356B
Application number: CN202410066538.6A
Authority: CN
Inventors: 于慧敏; 杜岩; 梁有向; 王苗苗; 陈博
Original assignee: Beijing Yanwei Technology Co ltd; Tsinghua University
Current assignee: Beijing Yanwei Technology Co ltd; Tsinghua University
Priority date: 2024-01-17
Filing date: 2024-01-17
Publication date: 2024-04-19
Anticipated expiration: 2044-01-17
Also published as: CN117586356A

Abstract

The application relates to the fields of biotechnology and genetic engineering, in particular to a polypeptide and application thereof. The length of the polypeptide is 20aa-50aa, and the amino acid sequence is as follows: m (X) _n-RX-（X）_m（A）_p-（X）_q -AXA (XXA, AXX), wherein n, M, p, q are any integer from 0 to 10, X is each occurrence, independently of the other, any amino acid; m (X) _n -RX is the N-terminal charged region; (X) _m（A）_p-（X）_q is an alanine-containing hydrophobic region; AXA (XXA, AXX) is an N-terminal charged region. The fusion expression of the polypeptide at the N end of the target protein can realize the secretory expression of the target protein in a host and can enhance the expression level of the target protein, namely the polypeptide has the dual functions of mediating the secretion of the target protein and enhancing the expression level of the target protein.

Description

Polypeptides and uses thereof

Technical Field

The application relates to the fields of biotechnology and genetic engineering, in particular to a polypeptide and application thereof.

Background

In the process of exogenously expressing target proteins, secretory expression has unique advantages and wide application scenarios compared with intracellular expression. For biocatalysis, some macromolecular substrates cannot cross cell membranes, and only secretion of enzymes outside cells can ensure that substrate molecules and enzymes are fully contacted, for example, catalytic decomposition reactions of lignin, cellulose and other polymers. Furthermore, the secretion strategy has significant advantages for heterologous protein production: firstly, secretion of target proteins can effectively avoid aggregation of intracellular soluble proteins to form inclusion bodies; secondly, it is more suitable to express some proteins which have toxic effects on cells; thirdly, because the bacterial cell is in a reduced state, some proteins with disulfide bonds cannot be folded in the cell correctly, and the problem can be effectively solved by secretion expression; finally, the secreted protein can effectively simplify the protein separation and purification steps, and greatly reduce the production cost. Therefore, it is important to increase the secretory expression ability of the target protein by the host.

Disclosure of Invention

Based on this, one or more embodiments of the present application provide a polypeptide, and fusion expression of the polypeptide at the N-terminus of a target protein can achieve secretory expression of the target protein in a host.

In a first aspect of embodiments of the application, there is provided a polypeptide having a length of 20aa to 50aa and an amino acid sequence as follows:

M（X）_n- RX-（X）_m（A）_p-（X）_q-AXA（XXA，AXX），

wherein,

N, m, p, q are any integer from 0 to 10, X is each occurrence, independently, any amino acid;

M (X) _n -RX is the N-terminal charged region;

(X) _m（A）_p-（X）_q is an alanine-containing hydrophobic region;

AXA (XXA, AXX) is an N-terminal charged region.

In some embodiments of the application, the amino acid sequence of the polypeptide is as set forth in any one of SEQ ID NO.1 to SEQ ID NO.50, or has one or more amino acid mutations, including insertions, deletions or substitutions, as compared to the sequence set forth in any one of SEQ ID NO.1 to SEQ ID NO.50, without altering the function of the polypeptide after mutation.

In a second aspect of embodiments of the application, there is provided a nucleic acid molecule comprising a polypeptide gene fragment encoding a polypeptide as described in the first aspect.

In some embodiments of the application, the nucleic acid molecule further comprises a gene segment of interest encoding a protein of interest and the polypeptide gene segment is linked to the 5' end of the gene segment of interest.

In some embodiments of the application, the gene fragment of interest comprises an enzyme.

In a third aspect of embodiments of the application, there is provided an expression vector comprising a nucleic acid molecule as described in the second aspect.

In a fourth aspect of embodiments of the present application, there is provided a genetically engineered bacterium comprising the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect.

In some embodiments of the application, the genetically engineered bacterium comprises a bacterium.

In some embodiments of the application, the bacteria include rhodococcus, bacillus subtilis, bacillus licheniformis, corynebacterium glutamicum, or escherichia coli.

In some embodiments of the application, the rhodococcus comprises rhodococcus erythropolis or rhodococcus turbidens.

In a fifth aspect of embodiments of the present application, there is provided a method for constructing a genetically engineered bacterium described in the fourth aspect, comprising the step of introducing the nucleic acid molecule described in the second aspect into a host.

In a sixth aspect of embodiments of the present application, there is provided a method for producing a target protein, comprising culturing the genetically engineered bacterium described in the fourth aspect; and isolating the protein of interest from the resulting culture.

Compared with the prior art, the implementation of the application has the following beneficial effects:

The embodiment of the application provides a polypeptide, which is fused and expressed at the N end of target protein to realize the secretory expression of the target protein in a host and enhance the expression level of the target protein, namely the polypeptide has the dual functions of mediating the secretion of the target protein and enhancing the expression level of the target protein.

Drawings

In order to more clearly illustrate the technical solution in the embodiments of the present application and to more fully understand the present application and its advantageous effects, the following brief description will be given with reference to the accompanying drawings, which are required to be used in the description of the embodiments. It is evident that the figures in the following description are only some embodiments of the application, from which other figures can be obtained without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram of a polypeptide structure;

FIG. 2 is a map of the pNV18.1-Pa2 plasmid;

FIG. 3 is a schematic diagram of plasmid expression vector construction with different polypeptide gene fragments;

FIG. 4 is a map of the pNV18.1-Pa2-BP6-eg plasmid;

FIG. 5 is a map of the pNV18.1-Pa2-BP7-eg plasmid;

FIG. 6 is a map of the pNV18.1-Pa2-BP8-eg plasmid;

FIG. 7 is a map of the pNV18.1-Pa2-BP10-mCherry plasmid;

FIG. 8 is a comparison of BP6 and BP6 mutant secretion effects;

FIG. 9 is a schematic representation of the appearance of a transparent "hydrolytic circle" around a recombinant strain obtained by ligating different polypeptide gene fragments to endoglucanases;

FIG. 10 is SDS-PAGE of supernatant of 48h fermentation broth of recombinant red rhodococcus secretion red fluorescent protein mCherry.

Detailed Description

The present application will be described in further detail with reference to the drawings, embodiments and examples. It should be understood that these embodiments and examples are provided solely for the purpose of illustrating the application and are not intended to limit the scope of the application in order that the present disclosure may be more thorough and complete. It will also be appreciated that the present application may be embodied in many different forms and is not limited to the embodiments and examples described herein, but may be modified or altered by persons skilled in the art without departing from the spirit of the application, and equivalents thereof are also intended to fall within the scope of the application. Furthermore, in the following description, numerous specific details are set forth in order to provide a more thorough understanding of the application, it being understood that the application may be practiced without one or more of these details.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing the embodiments and examples only and is not intended to be limiting of the invention.

Terminology

Unless otherwise indicated or contradicted, terms or phrases used herein have the following meanings:

The term "and/or," "and/or," as used herein, includes any one of two or more of the listed items in relation to each other, as well as any and all combinations of the listed items in relation to each other, including any two of the listed items in relation to each other, any more of the listed items in relation to each other, or all combinations of the listed items in relation to each other. It should be noted that, when at least three items are connected by a combination of at least two conjunctions selected from the group consisting of "and/or", "and/or", it should be understood that, in the present application, the technical solutions include technical solutions that all use "logical and" connection, and also include technical solutions that all use "logical or" connection. For example, "a and/or B" includes three parallel schemes A, B and a+b. For another example, the technical schemes of "a, and/or B, and/or C, and/or D" include any one of A, B, C, D (i.e., the technical schemes of all "logical or" connections), also include any and all combinations of A, B, C, D, i.e., the combinations of any two or three of A, B, C, D, and also include four combinations of A, B, C, D (i.e., the technical schemes of all "logical and" connections).

The terms "plurality", "plural", "multiple", and the like in the present invention refer to, unless otherwise specified, an index of 2 or more in number. For example, "one or more" means one kind or two or more kinds.

As used herein, "a combination thereof," "any combination thereof," and the like include all suitable combinations of any two or more of the listed items.

The "suitable" in the "suitable combination manner", "suitable manner", "any suitable manner" and the like herein refers to the fact that the technical scheme of the present invention can be implemented, the technical problem of the present invention is solved, and the technical effect expected by the present invention is achieved.

Herein, "preferred", "better", "preferred" are merely to describe better embodiments or examples, and it should be understood that they do not limit the scope of the invention.

In the present invention, "further", "still further", "particularly" and the like are used for descriptive purposes to indicate differences in content but should not be construed as limiting the scope of the invention.

In the present invention, "optional" means optional or not, that is, means any one selected from two parallel schemes of "with" or "without". If multiple "alternatives" occur in a technical solution, if no particular description exists and there is no contradiction or mutual constraint, then each "alternative" is independent.

In the present invention, the terms "first", "second", "third", "fourth", etc. are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or quantity, nor as implying an importance or quantity of a technical feature being indicated. Moreover, the terms "first," "second," "third," "fourth," and the like are used for non-exhaustive list description purposes only, and are not to be construed as limiting the number of closed forms.

In the invention, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.

In the present invention, a numerical range (i.e., a numerical range) is referred to, and optional numerical distributions are considered to be continuous within the numerical range and include two numerical endpoints (i.e., a minimum value and a maximum value) of the numerical range and each numerical value between the two numerical endpoints unless otherwise specified. Unless otherwise indicated, when a numerical range merely refers to integers within the numerical range, both end integers of the numerical range are included, as well as each integer between the two ends, herein, each integer is recited directly, such as t is an integer selected from 1-10, and t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Further, when a plurality of range description features or characteristics are provided, these ranges may be combined. In other words, unless otherwise indicated, the ranges disclosed herein are to be understood to include any and all subranges subsumed therein.

The temperature parameter in the present invention is not particularly limited, and may be a constant temperature treatment or may vary within a predetermined temperature range. It should be appreciated that the constant temperature process described allows the temperature to fluctuate within the accuracy of the instrument control. Allows for fluctuations within a range such as + -5 ℃, + -4 ℃, + -3 ℃, + -2 ℃, + -1 ℃.

In the present invention,% (w/w) and wt% each represent weight percent,% (v/v) represents volume percent, and% (w/v) represents mass volume percent.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Unless otherwise indicated to the contrary by the intent and/or technical aspects of the present application, all references to which this application pertains are incorporated by reference in their entirety for all purposes. When reference is made to a cited document in the present application, the definitions of the relevant technical features, terms, nouns, phrases, etc. in the cited document are also incorporated. In the case of the cited documents, examples and preferred modes of the cited relevant technical features are also incorporated into the present application by reference, but are not limited to being able to implement the present application. It should be understood that when a reference is made to the description of the application in conflict with the description, the application is modified in light of or adaptive to the description of the application.

The N-terminal sequence of the target protein is particularly important in the protein secretion process. To achieve secretory expression of a protein, a polypeptide sequence having a secretory function must be fused to the N-terminus of the sequence encoding the protein of interest. Recent researches show that the N-terminal of the protein also affects the factors such as biosynthesis, folding dynamics, stability and the like of the target protein. According to the embodiment of the application, the polypeptide is fused and expressed at the N end of the target protein, so that the secretory expression of the target protein is realized.

First aspect of embodiments of the application

The embodiment of the application provides a polypeptide, the length of which is 20aa-50aa, and the amino acid sequence is as follows:

M（X）_n- RX-（X）_m（A）_p-（X）_q-AXA（XXA，AXX），

wherein,

m (X) _n -RX is the N-terminal charged region (i.e., region 1);

(X) _m（A）_p-（X）_q is an alanine-containing hydrophobic region (i.e., region 2);

AXA (XXA, AXX) is the N-terminal charged region (i.e. region 3).

In the embodiment of the present application, "AXA (XXA, AXX)" means that AXA may be used, XXA may be used, or AXX may be used.

From the above amino acid sequences it will be appreciated that region 1 is an N-terminal charged region, starting with methionine M and ending with RX (e.g.RR), in region 2, (A) _p is between (X) _m and (X) _q, region 3 is a C-terminal charged region and ends with three signature sequences AXA or XXA, AXX.

Alternatively, the polypeptide has an amino acid sequence as set forth in any one of SEQ ID NO.1 to SEQ ID NO.50, or has one or more amino acid mutations including insertions, deletions or substitutions as compared to the sequence set forth in any one of SEQ ID NO.1 to SEQ ID NO.50, and the function of the polypeptide is unchanged after the mutation. Alternatively, the number of amino acid mutations is from 1 to 5 (e.g., 1,2,3, 4, 5). Alternatively, the substitutions comprise conservative substitutions, in particular conservative substitutions of amino acid X.

SEQ ID NO.1 (35aa)：MRISATAVRAAAVAVASRAAVAVVLAVVVATPAAA。

SEQ ID NO.2 (29aa)：MSRRTALRAGGAVLGAAALAAVCASPASA。

SEQ ID NO.3 (41aa)：MNPIALPPRPHLSRRSFLGLGAAAGAVLLGACSTSGETAPA。

SEQ ID NO.4 (36aa)：MTIRTEPLRLSRRGFLAAGAGALAATALGGWTPVHA。

SEQ ID NO.5 (33aa)：MRRPAGAIDRRTMLAGTAVLAGAALIGAAPARA。

SEQ ID NO.6 (38aa)：MQTGTSRGMKRLAGGAALAAAAAATVAVTMPATASAAT。

SEQ ID NO.7 (48aa)：

MVSSGHAVPPAARDGVSFVKRTRALAAASLVGAAVTLIAFAGPAAANP。

SEQ ID NO.8 (50aa)：

MHTSSNESGHMGKSGIGFSRNKHWSSRVAVALTGAVVSGTALVGAAQAAP。

SEQ ID NO.9 (42aa)：

MIVTATKPSHGWLRGVVRLMVAVVILPLAFVLVGGGTASADP。

SEQ ID NO.10 (45aa)：

MTSQRRRTMVNRTAAGRYGVRFALAVALTAAIPCLGVQASASADP。

SEQ ID NO.11 (27aa)：MPHRRPKPSIVLGAVAALAVASPFAVY。

SEQ ID NO.12 (39aa)：MRFVPRLSQRLRRRVLGVGAAALVLPVAAGVAGSTAALA。

SEQ ID NO.13 (38aa)：MSGRHRKPTNTGRTVAKFALTSAVLGVTGVAFSGTANA。

SEQ ID NO.14 (25aa)：MIRRALTTAALAAAATLVLAPTATA。

SEQ ID NO.15 (33aa)：MASQISKRTVRRAVVAGALALGAVTVSAGPALA。

SEQ ID NO.16 (26aa)：MKFRKLAAVAAMTIAAVGLTAGTSYA。

SEQ ID NO.17 (28aa)：MQSVLKKTARTVAGVGALALCVPGTASA。

SEQ ID NO.18 (39aa)：MALDWAVRNKRIAAAAVAPVALGAAAVIAVAVSEPSAPS。

SEQ ID NO.19 (35aa)：MTRGTKTVARAAAAAVAAVGIGAGLAVAAPAVASA。

SEQ ID NO.20 (38aa)：MRYSSASRAAARTTVRRVVVAAASALLLAGPLAATAHA。

SEQ ID NO.21 (36aa)：MSNKHTSGLRRGARIGGVAAAAAVALGFLSAGAANA。

SEQ ID NO.22 (29aa)：MRRSFARRAAVYGSAALMLFGPAAAIAQA。

SEQ ID NO.23 (26aa)：MDLEQFPVSRRSVLLGAGVAAVAATA。

SEQ ID NO.24 (26aa)：MDLEQFPVSRRSVLLGAGVAAVAAAA。

SEQ ID NO.25 (31aa)：MNTRRRTVAALAAGVAIGIAGFGTAVATAQA。

SEQ ID NO.26 (34aa)：MTMFPPRRRTLLALATTAALAAGATACGTGTTPA。

SEQ ID NO.27 (31aa)：MSSRTSRRRFLGTAAGGVVGLGLLPAARAAA。

SEQ ID NO.28 (42aa)：MRKFETGKNAGGTHAARRHVRRGSAAAAALLACLIAPAPSAA。

SEQ ID NO.29 (28aa)：MRIRHTLTVAVAAVLAVAGCGGQDAPRA。

SEQ ID NO.30 (29aa)：MMQMSRRSFLLGASAAAGAMALGGLTGCA。

SEQ ID NO.31 (29aa)：MSRRTALRAGGAVLGAAALAAVSASPASA。

SEQ ID NO.32 (33aa)：MTKMLRDRRILAAAAALAVLALAALLLPHPSVA。

SEQ ID NO.33 (36aa)：MSPISCPSPLTSARLSRRRFLTVAAMASAAVPILSA。

SEQ ID NO.34 (33aa)：MLGRRRFLALGSLGAGSLGLSALGFGAAGCATA。

SEQ ID NO.35 (46aa)：

MLDRAYREIEDGRARFGRRSFLAALGVSAAGLGVSAAGLGAATAAA。

SEQ ID NO.36 (40aa)：MGMRRRPLSVAAAIAAGVAAAIAAGVAAAVAAAACGSAPA。

SEQ ID NO.37 (39aa)：MRPPGAVLGRRRFLALGSLGAGALGMSALGFGAAGCATA。

SEQ ID NO.38 (41aa)：MNPVTLSALPHLSRRSFLGLGAAAGAVLLGACSTSGETAPA。

SEQ ID NO.39 (29aa)：MNGARRIAGALAGIAAAVAAVTGAATVPA。

SEQ ID NO.40 (37aa)：MPVLTVPTRRLFAAGLAALALLAASCASSGGSEPASA。

SEQ ID NO.41 (27aa)：MFDTRLCRRTFLTALGALAVAPAPAHA。

SEQ ID NO.42 (35aa)：MSSGWGLLVFDTRFSRRTFLTALGALAVTPAPAHA。

SEQ ID NO.43 (38aa)：MNIDRRGFLGLTGLVAASAALAACAGTGSSGSSESASA。

SEQ ID NO.44 (27aa)：MFDTRLSRRTFLTALGALAVAPAPAHA。

SEQ ID NO.45 (38aa)：MNIDRRRFLGLTGVVAASAALAACAGTGSSGPSESAAA。

SEQ ID NO.46 (30aa)：MVVDRTKRSLRALMIGVLLAVFSVAVPAGA。

SEQ ID NO.47 (34aa)：MGIKSRGFKAARNAAVSGAAILGLVLGATGTAQA。

SEQ ID NO.48 (21aa)：MITACALRAAAALTVPSQAVA。

SEQ ID NO.49 (36aa)：MKTSITKGLRRGLQAAGVGATIAVAMGFASVGAANA。

SEQ ID NO.50 (41aa)：MFEKKGRPNSRGRAGRWSGRVVTALVGVGAATLTMTGPAAA。

Alternatively, the sequence of the polypeptide is shown as SEQ ID NO. 76.

The polypeptides shown in SEQ ID No.1 to SEQ ID No.50 and SEQ ID No.76 are represented by BP1 to BP 51.

The polypeptides provided by the embodiment of the application can exist independently, and the polypeptides with different sequences can also be combined into a polypeptide library.

In the art, mutations of the above type generally do not alter the function of the polypeptide, in particular substitutions using amino acids of similar properties, i.e. conservative substitutions.

These conservatively substituted polypeptides are preferably generated by amino acid substitutions according to Table A.

Table A

In a second aspect of embodiments of the application

Embodiments of the present application provide a nucleic acid molecule comprising a polypeptide gene fragment encoding a polypeptide as described in the first aspect.

Optionally, the nucleic acid molecule further comprises a gene fragment of interest encoding a protein of interest and the polypeptide gene fragment is linked to the 5' end of the gene fragment of interest.

The kind of the target protein encoded by the target gene fragment is not particularly limited in the embodiment of the application, and the target protein includes, but is not limited to, enzymes, such as monosubunit enzymes or polymerase composed of homosubunits, and may be endoglucanase, exoglucanase, beta-glucosidase, red fluorescent protein, lipase, nattokinase, and the like, and mutants of these enzymes. The amino acid sequence of the target protein can be shown as the sequence shown in any one of SEQ ID NO. 51-SEQ ID NO.55, or can be a sequence with high homology (such as not less than 90%) with the sequence shown in any one of SEQ ID NO. 51-SEQ ID NO. 55.

SEQ ID NO.51 (885aa)：

MKKRLVKKVAMLIAIVLVLSSSIGQAFALVGAGDLIRNHTFDNRVGLPWHVVESYPAKASFEITSDGKYKITAQKIGEAGKGERWDIQFRHRGLALQQGHTYTVKFTVTASRACKIYPKIGDQGDPYDEYWNMNQQWNFLELQANTPKTVTQTFTQTKGDKKNVEFAFHLAPDKTTSEAQNPASFQPITYTFDEIYIQDPQFAGYTEDPPEPTNVVRLNQVGFYPNADKIATVATSSTTPINWQLVNSTGAAVLTGKSTVKGADRASGDNVHIIDFSSYTTPGTDYKIVTDVSVTKAGDNESMKFNIGDDLFTQMKYDSMKYFYHNRSAIPIQMPYCDQSQWARPAGHTTDILAPDPTKDYKANYTLDVTGGWYDAGDHGKYVVNGGIATWTVMNAYERALHMGGDTSVAPFKDGSLNIPESGNGYPDILDEARYNMKTLLNMQVPAGNELAGMAHHKAHDERWTALAVRPDQDTMKRWLQPPSTAATLNLAAIAAQSSRLWKQFDSAFATKCLTAAETAWDAAVAHPEIYATMEQGAGGGAYGDNYVLDDFYWAACELYATTGSDKYLNYIKSSKHYLEMPTELTGGENTGITGAFDWGCTAGMGTITLALVPTKLPAADVATAKANIQAAADKFISISKAQGYGVPLEEKVISSPFDASVVKGFQWGSNSFVINEAIVMSYAYEFSDVNGTKNNKYINGALTAMDYLLGRNPNIQSYITGYGDNPLENPHHRFWAYQADNTFPKPPPGCLSGGPNSGLQDPWVKGSGWQPGERPAEKCFMDNIESWSTNEITINWNAPLVWISAYLDEKGPEIGGSVTPPTNLGDVNGDGNKDALDFAALKKALLSQDTSTINVANADINKDGSIDAVDFALLKSFLLGKITL.

SEQ ID NO.52 (475aa)：

MKKTTAFLLCFLMIFTALLPMQNANAYDASLIPNLQIPQKNIPNNDGMNFVKGLRLGWNLGNTFDAFNGTNITNELDYETSWSGIKTTKQMIDAIKQKGFNTVRIPVSWHPHVSGSDYKISDVWMNRVQEVVNYCIDNKMYVILNTHHDVDKVKGYFPSSQYMASSKKYITSVWAQIAARFANYDEHLIFEGMNEPRLVGHANEWWPELTNSDVVDSINCINQLNQDFVNTVRATGGKNASRYLMCPGYVASPDGATNDYFRMPNDISGNNNKIIVSVHAYCPWNFAGLAMADGGTNAWNINDSKDQSEVTWFMDNIYNKYTSRGIPVIIGECGAVDKNNLKTRVEYMSYYVAQAKARGILCILWDNNNFSGTGELFGFFDRRSCQFKFPEIIDGMVKYAFEAKTDPDPVIVYGDYNNDGNVDALDFAGLKKYIMAADHAYVKNLDVNLDNEVNAFDLAILKKYLLGMVSKLPSN.

SEQ ID NO.53 (711aa)：

MQYDQIDKKIDELLSMMTLEEKAGMCHGAGLFRTAGVPRLGIPPLVFSDGPMGIRNEFADDNWNTVGGNTDFVTYLPANTALAATFNRTLAESLGEVLGCEARGRGKDVILAPGVNIIRTPLCGRNYEYFSEDPILTAELAASFIKGVQRFDVAACVKHFAANNQETERLAVSAEVDELTLRELYFPAFEASVRAGVLTVMTAYNRLNGTFCSHSRQLITEILREEWGFNGVVVSDWGAVHDTESPAIAGLDIEMNVTSNFNEYFFAKPLINAIKDGKIPERMLDDKVRRILRLMFRLNMFSKDRKRGGFNLPQHQQAVLDAAKESFVLLKNDREVLPLNADGIKTVAVIGSNADKKHSSGGDSAAVKALYEVTPLSGIVMRLASGAKVTYYPGCPDETHYKEEFHIPSNADEKTRADIEEKARAADEDYRKIQMRLEDEAIQAAKTADAVIFIGGNGHEQESEGRDRPDMALPYEQDKLLSRVLDANPNTVVVIISGSPVNMSGWIDKAPTVMQGFFSGMHGGTALAAVLFGDENPSGHLPFTIPLKEEETGASALGEYPGGETVCYSEGLFVGYRYHDAFNIPPLFPFGYGLSYTTFSLANESFRRLPCVGTEYEIHVDITNSGNRPGAQSVQLYVEPEKKDGSPIRTLKGFEKTYLNPDETKTITFKLDERTFSEFRPHEGWVFVPGNYTIHIGTSSRELPIAIALAL.

SEQ ID NO.54 (236aa)

MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK.

SEQ ID NO.55 (270aa)：

MSINGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATVLLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPFTSVLDHLSYFGINTGLCT.

Third aspect of embodiments of the application

Embodiments of the present application provide an expression vector comprising a nucleic acid molecule as described in the second aspect.

Fourth aspect of embodiments of the application

The embodiment of the application provides a genetically engineered bacterium, which comprises the nucleic acid molecule in the second aspect or the expression vector in the third aspect.

The embodiment of the application does not particularly limit the type of the genetically engineered bacterium, and bacteria can be selected for example. The type of bacteria is not particularly limited, and may be, for example, rhodococcus (e.g., rhodococcus rhodochrous (Rhodococcus ruber) or rhodococcus cloudus (Rhodococcus opacus)), bacillus subtilis, bacillus licheniformis, corynebacterium glutamicum, escherichia coli, and the like. Wherein the rhodococcus is an aerobic gram-positive bacterium, and is a non-model strain. As the rhodococcus has higher organic solvent tolerance and rich nitrile and aromatic compound metabolizing enzyme systems, the rhodococcus has wide application in the fields of biocatalysis, environmental remediation, biosynthesis, wood degradation utilization and the like. For example: in the field of biocatalysis, rhodococcus has been successfully used in the biological production of acrylamide, acrylic acid and other compounds; in the field of biosynthesis, rhodococcus can synthesize a number of high value-added products such as biosurfactants, carotenoids, antibacterial agents, triacylglycerols, and polyhydroxyalkanoates. In addition, rhodococcus is also a highly efficient protein expression host, for example in rhodococcus roseus (Rhodococcus rhodochrous), nitrile hydratase can account for about 45% of the total amount of soluble protein. The application fields are all to achieve the purposes of biocatalysis, biosynthesis and the like by expressing target proteins in rhodococcus cells. In view of various characteristics and important potential application values of the rhodococcus, the improvement of the secretion expression capacity of the rhodococcus to target proteins plays an important role in widening the application field of the rhodococcus and developing a rhodococcus catalysis and synthesis platform.

The genetically engineered bacterium of the embodiment of the application may comprise the vector, or may integrate the nucleic acid molecule in the genome.

Fifth aspect of embodiments of the application

An embodiment of the present application provides a method for constructing a genetically engineered bacterium described in the fourth aspect, comprising the step of introducing the nucleic acid molecule described in the second aspect into a host to construct a genetically engineered bacterium.

The construction method of the embodiment of the application comprises the following steps: the nucleic acid molecules are connected into an escherichia coli-nocardia/rhodococcus shuttle plasmid to construct a recombinant plasmid vector, and the recombinant plasmid vector is transformed into a host cell. Further, the shuttle plasmid may be one of pNV, pnv18.1, pNV, pRC, pΦc31 plasmid or a derivative thereof.

The inventor obtains 50 polypeptides with mediated target protein secretory expression through multiple screening by analyzing and identifying rhodococcus secretory protein groups and analyzing and identifying rhodococcus source secretory protein sequences recorded in a database. Embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples, in which specific conditions are not noted, are preferably referred to the guidelines given in the present invention, and may be according to the experimental manual or conventional conditions in the art, the conditions suggested by the manufacturer, or the experimental methods known in the art.

In the specific examples described below, the measurement parameters relating to the raw material components, unless otherwise specified, may have fine deviations within the accuracy of weighing. Temperature and time parameters are involved, allowing acceptable deviations from instrument testing accuracy or operational accuracy.

In the following specific examples, the structure of the polypeptide involved is shown in FIG. 1, and a schematic diagram of recombinant vector construction using pNV18.1-Pa2 (see FIG. 2) is shown in FIG. 3.

EXAMPLE 1 construction of recombinant vector pNV-18.1-Pa2-BP-eg carrying exogenous endoglucanase Gene

1. Selecting an endoglucanase gene from clostridium cellulolyticum, carrying out codon optimization on a rhodococcus rhodochrous host, and the nucleotide sequence of the optimized gene is shown as SEQ ID NO. 56. The gene synthesis work is entrusted to be completed by Beijing Liuhua big gene technology Co. SEQ ID NO.56 is as follows:

ATGAAGAAGACCACCGCCTTCCTGCTGTGCTTCCTGATGATCTTCACCGCCCTGCTGCCGATGCAGAACGCCAATGCCTATGATGCCTCGCTGATTCCGAATCTGCAGATCCCGCAAAAGAACATCCCGAACAACGACGGCATGAACTTCGTCAAGGGCCTGCGCTTAGGCTGGAACCTGGGCAATACCTTTGACGCCTTTAACGGCACCAACATCACCAACGAGCTGGACTACGAGACCTCGTGGTCGGGCATTAAGACCACCAAACAGATGATCGACGCCATCAAGCAGAAGGGCTTCAACACCGTCCGCATCCCGGTCTCGTGGCATCCGCATGTCTCGGGCTCGGATTATAAGATTTCGGACGTCTGGATGAACCGCGTCCAGGAGGTCGTTAATTACTGCATCGACAACAAGATGTACGTCATCCTGAACACCCACCACGACGTCGACAAGGTCAAGGGCTATTTCCCGTCGTCGCAGTACATGGCCTCGTCGAAAAAGTACATCACCTCGGTCTGGGCCCAGATCGCCGCACGTTTTGCCAATTATGATGAACATCTGATCTTCGAGGGCATGAACGAGCCGCGCCTGGTCGGCCATGCCAATGAATGGTGGCCGGAACTGACCAATTCGGACGTCGTCGACTCGATTAACTGCATCAACCAGCTGAACCAGGACTTCGTCAACACCGTCCGCGCCACCGGCGGTAAAAATGCCTCGCGCTATCTGATGTGCCCGGGCTATGTTGCCTCGCCGGATGGCGCCACCAATGATTATTTCCGCATGCCGAACGACATCTCGGGCAACAACAACAAGATCATCGTCTCGGTCCACGCCTACTGCCCGTGGAATTTTGCCGGCCTGGCCATGGCCGATGGCGGCACCAACGCCTGGAACATTAACGATTCGAAGGACCAGTCGGAGGTCACCTGGTTCATGGACAACATCTACAACAAGTACACCTCGCGCGGCATCCCGGTCATTATTGGCGAATGCGGCGCCGTCGATAAAAATAATCTGAAGACCCGCGTCGAGTACATGTCGTACTACGTCGCCCAGGCCAAGGCCCGCGGTATACTGTGTATTCTGTGGGACAATAACAACTTCTCGGGCACCGGCGAGCTGTTCGGCTTTTTTGATCGCCGCTCGTGTCAGTTCAAGTTCCCGGAGATAATCGACGGCATGGTCAAGTACGCCTTCGAGGCCAAAACCGACCCGGACCCAGTTATTGTTTATGGCGATTACAATAACGACGGCAACGTCGACGCCCTGGACTTCGCAGGCTTGAAAAAGTATATCATGGCCGCCGACCACGCCTACGTCAAGAATCTGGATGTCAACCTGGACAACGAGGTCAACGCCTTCGACCTGGCCATTCTGAAGAAGTACCTGCTGGGCATGGTCTCGAAGCTGCCGTCGAATTGA.

2. The endoglucanase gene eg is amplified by PCR reaction by taking the gene sequence synthesized by the steps as a template and taking the eg6-F/eg6-R, eg7-F/eg7-R, eg-F/eg 8-R as a primer, and then the endoglucanase gene eg is recovered and purified. The genes of polypeptides BP6 (SEQ ID NO. 6), BP7 (SEQ ID NO. 7) and BP8 (SEQ ID NO. 8) are amplified, recovered and purified by taking BP6-F/BP6-R, BP-F/BP 7-R, BP-F/BP 8-R as primers and taking rhodococcus genomic DNA as a template. The plasmid backbone pNV18.1-Pa2 (shown in FIG. 2) was digested with restriction enzymes XbaI and KpnI, and the linearized plasmid was recovered and purified. Three connection systems were constructed using the Gibson Assembly kit (Clonesmarter brand, available from Zhongmeitai and Biotechnology (Beijing) Inc.), respectively: linearization vector +bp6+eg; linearization vector +bp7+eg; linearized vector +bp8+eg.

3. The ligation product of step2 was transformed into E.coli trans10 competent cells (purchased from Beijing full gold biotechnology Co., ltd.) by chemical transformation, and the procedure was as follows, 10. Mu.L of the ligation system was added to 100. Mu.L of competent cells, placed on ice for 30 minutes, heat shock at 42℃for 45 seconds, then placed on ice for 2 minutes, then 900. Mu.L of non-resistant SOC medium was added, placed on a shaker for 60 minutes, resuscitated under 200rpm,37℃after resuscitated, 200. Mu.L of bacterial liquid was spread on LB medium solid plates containing 50. Mu.g/mL kanamycin, positive clones were selected, and PCR and sequencing were performed to obtain recombinant vectors pNV18.1-Pa2-BP6-eg (as shown in FIG. 4), pNV18.1-Pa2-BP7-eg (as shown in FIG. 5), and pNV18.1-Pa2-BP8-eg (as shown in FIG. 6).

SOC medium: 20g/L peptone, 5g/L yeast powder, 0.5g/L sodium chloride, 2.5mM potassium chloride, 10mM magnesium chloride, 20mM glucose and water.

LB solid medium: peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L, pH=7.0, and solid plate culture medium containing agar 1.5% -2% and water.

Primers were synthesized by the biological technology limited of the safe and up to stora and used by dilution to 10 μm with sterile water. The PCR amplification was performed using high-fidelity DNA polymerase Phanta Max Master X2X mix from Nanjinouzan.

The sequences (5 '-3') of the primers are as follows:

eg6-F（SEQ ID NO.58）：ACCGCCAGTGCCGCCACATATGATGCCTCGCTGATTC；

eg6-R（SEQ ID NO.59）：

ATGATTACGAATTCGAGCTCGGTACCTCAATTCGACGGCAGCTTCG；

SP6-F（SEQ ID NO.60）：GCGAGTCACTAAGGAGTCTAGAATGCAAACCGGCACCTCG；

BP6-R（SEQ ID NO.61）：ATCATATGTGGCGGCACTGGCGG；

BP7-F（SEQ ID NO.62）：

GGCGAGTCACTAAGGAGTCTAGAATGGTCTCGTCGGGCCATG；

BP7-R（SEQ ID NO.63）：ATCATACGGATTGGCTGCGGCTG；

eg7-F（SEQ ID NO.64）：CAGCCGCAGCCAATCCGTATGATGCCTCGCTGATTC；

eg7-R（SEQ ID NO.65）：

TGATTACGAATTCGAGCTCGGTACCTCAATTCGACGGCAGCTTCGAGACC；

BP8-F（SEQ ID NO.66）：

GGCGAGTCACTAAGGAGTCTAGAATGCATACCTCGTCGAACG；

BP8-R（SEQ ID NO.67）：AGCGAGGCATCATACGGGGCGGCTTGGGCGG；

eg8-F（SEQ ID NO.68）：AAGCCGCCCCGTATGATGCCTCGCTG；

eg8-R（SEQ ID NO.69）：GAATTCGAGCTCGGTACCTCAATTCGACGGCAGC。

And (3) PCR reaction:

the PCR reaction system is as follows: 1 μl of template, 2 μl of upstream primer, 2 μl of downstream primer, phanta Max Master Mix μl of sterile water, 20 μl of sterile water, and a total volume of 50 μl;

The PCR reaction conditions were: 3min at 95 ℃;95 ℃ 30s,55 ℃ 15s,72 ℃ 2min,35 cycles; 5 min at 72 ℃;4℃forever.

EXAMPLE 2 construction of recombinant plasmid pNV-18.1-Pa2-BP6-mut-eg carrying the BP6 Point mutant

1. The last amino acid of BP6 polypeptide is subjected to T38A mutation, and the construction process is as follows:

The recombinant plasmid pNV18.1-Pa2-BP6-eg constructed in example 1 is used as a template, BP6-mut-F/BP6-mut-R is used as a primer to carry out PCR for linearization treatment on the plasmid, and the PCR product is recovered and purified. The linearized vector was ligated using the Gibson Assembly kit (Clonesmarter brand, available from Zhongmeitai and Biotechnology (Beijing) Inc.).

2. The ligation product was transformed into E.coli trans10 competent cells by chemical transformation (purchased from Beijing full gold Biotechnology Co., ltd., see example 1 for specific procedures) to obtain recombinant vector pNV18.1-Pa2-BP6-mut-eg.

The PCR reaction system is as follows: 1. Mu.L (1 ng/. Mu.L) of template, 2. Mu.L of upstream primer, 2. Mu.L of downstream primer, phanta Max Master Mix. Mu.L, 20. Mu.L of sterile water and a total volume of 50. Mu.L;

the PCR reaction conditions were: 3min at 95 ℃;95 ℃ for 30s,55 ℃ for 15s,72 ℃ for 6min,35 cycles; 5 min at 72 ℃;4℃forever.

BP6-mut-F（SEQ ID NO.70）：ACCGCCAGTGCCGCCGCGTATGATGCCTCGCTGATTCCG

BP6-mut-R（SEQ ID NO.71）：GCATCATACGCGGCGGCACTGGCGGTGGCCGGCATG

EXAMPLE 3 construction of recombinant vector pNV-18.1-Pa2-BP10-mCherry carrying red fluorescent protein Gene mCherry

1. Selecting red fluorescent protein mCherry gene, carrying out codon optimization on a red rhodococcus host, and the nucleotide sequence of the gene after optimization is shown as SEQ ID NO. 57. The gene synthesis work is entrusted to be completed by Beijing Liuhua big gene technology Co. SEQ ID NO.57 is as follows:

ATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTGA.

2. And respectively using mCherry-F/mCherry-R as a primer, using the gene sequence synthesized in the steps as a template, amplifying the mCherry gene fragment by PCR reaction, and recovering and purifying. The BP10-F/BP10-R is used as a primer, the rhodococcus erythropolis genome DNA is used as a template, and the gene sequence of the polypeptide sequence BP10 is amplified, recovered and purified. The plasmid backbone pNV18.1-Pa2 was double digested with restriction enzymes XbaI and KpnI, and the linearized plasmid was recovered and purified. The connection was performed using a Gibson Assembly kit (Clonesmarter brand, available from Zhongmeitai and Biotechnology (Beijing) Co., ltd.) with the connection system: linearized vector +bp10+mcherry.

3. The ligation product was transformed into E.coli trans10 competent cells by chemical transformation (purchased from Beijing full gold Biotechnology Co., ltd., specific procedures are shown in example 1. Recombinant vector pNV18.1-Pa2-BP10-mCherry was obtained (as shown in FIG. 7).

The formulation of the culture medium is shown in example 1.

The primer sequences (5 '-3') were as follows:

BP10-F（SEQ ID NO.72）：

GCCCGGCGAGTCACTAAGGAGTCTAGAATGACATCGCAACGCCGCCG；

BP10-R（SEQ ID NO.73）：TCGCCCTTGCTCACCATCGGATCGGCCGAGGCCGAGG；

mCherry-F（SEQ ID NO.74）：GATCCGATGGTGAGCAAGGGCGAGGAG；

mCherry-R（SEQ ID NO.75）：

ATGATTACGAATTCGAGCTCGGTACCTCACTTGTACAGCTCGTCC。

EXAMPLE 4 construction of recombinant Rhodococcus encoding exogenous endoglucanase

The recombinant plasmids constructed in example 1 and example 2 were transferred into Rhodococcus R. ruber THdAdN (construction method is detailed in China patent application CN 105420154A) by electrotransformation, respectively, to obtain recombinant Rhodococcus R.ruber BP6-EG, R.ruber BP6-mut-EG, R.ruber BP7-EG and R.ruber BP8-EG. The specific operation method is as follows: adding 10 mu L of the recombinant plasmid into 80 mu L of R. ruber THdAdN competent cells, uniformly mixing, performing ice bath for 10 minutes, and transferring into a 1mm electric rotating cup; adjusting the voltage of the electroporation device to 1.25 kV, loading the electroporation device with the electric rotating cup, and pressing an electric shock key; immediately after the end of the shock, 900 μl of LBHIS medium was added to the cuvette, the cells resuspended, and transferred to a 1.5mL centrifuge tube; resuscitating at 200rpm at 28℃for 2.5 hours; after resuscitating, centrifuging at 13000rpm at high speed, removing 800 mu L of supernatant, re-suspending the centrifuged cells, coating on a rhodococcus solid culture medium (kanamycin 25 mu g/mL), placing in a 28 ℃ incubator, culturing for 48-60h in an inverted manner, and selecting positive clones to obtain the recombinant rhodococcus capable of secreting and expressing exogenous endoglucanase.

LBHIS medium: 5g/L peptone, 5g/L sodium chloride, 2.5g/L yeast powder, 18.5g/L brain-heart extract, 91g/L sorbitol and water.

Rhodococcus solid medium: 10g/L glucose, 3g/L yeast extract, 1g/L sodium chloride, 2g/L dipotassium phosphate trihydrate, 0.2g/L magnesium sulfate heptahydrate, 15 g/L agar, water and natural pH.

EXAMPLE 5 construction of recombinant Rhodococcus encoding red fluorescent protein

The recombinant plasmid constructed in example 3 was transferred into rhodococcus R. ruber THdAdN (construction method is shown in Chinese patent application CN 105420154A) by electrotransformation to obtain recombinant rhodococcus R.ruber BP10-mCherry. The specific operation method is the same as in example 4. And selecting positive clones to obtain the recombinant rhodococcus capable of secreting red fluorescent protein mCherry.

Example 6 determination of recombinant Rhodococcus extracellular endoglucanase Activity

The recombinant rhodococcus extracellular endoglucanase activity was determined using congo red hydrolysis circle method. The specific operation method is as follows: the recombinant rhodococcus constructed in example 4 was streaked on rhodococcus solid medium, single colony was picked and inoculated on rhodococcus solid fermentation medium containing sodium carboxymethylcellulose (CMC-Na, 1%) and placed in a 28℃incubator for 48 hours, after which 20mL of Congo red dye was added for 30 minutes, and then decolorized with 1M NaCl solution for 30 minutes. The size of the extracellular endoglucanase activity was characterized by measuring the diameter of the hydrolysis circle in two perpendicular directions.

Rhodococcus solid fermentation medium: 30g/L glucose, 7-8g/L yeast extract, 10g/L urea, 0.866g/L monopotassium phosphate, 2.28g/L dipotassium phosphate trihydrate, 1g/L magnesium sulfate heptahydrate, 1g/L monosodium glutamate, 10g/L sodium carboxymethyl cellulose, 15g/L agar, water and regulating the pH to 7.5.

Other fusion expression recombinant vectors were constructed according to the method of example 1, and recombinant rhodococcus was constructed according to the method of example 2, and endoglucanase activity was detected. The results are shown in tables 1,2, fig. 8 and fig. 9.

TABLE 1

TABLE 2

The amino acid sequence of BP51 (BP 6-mut, T38A) is: MQTGTSRGMKRLAGGAALAAAAAA TVAVTMPATASAAA (SEQ ID NO. 76).

EXAMPLE 7 recombinant Rhodococcus extracellular Red fluorescent protein denatured Polyacrylamide gel electrophoresis

Plate activating the recombinant rhodococcus constructed in example 5, picking single colony, inoculating in rhodococcus seed culture medium, culturing at 28 deg.c and 200rpm for 48-60 hr; inoculated in 10% inoculum size into rhodococcus fermentation medium, fermented at 28 ℃ at 200rpm for 48 hours. After the fermentation, a certain volume of fermentation broth is taken, centrifuged at 13000rpm for 10 minutes to remove thalli, and the supernatant of the fermentation broth is collected for SDS-PAGE identification.

Rhodococcus seed culture medium: glucose 20g/L, yeast extract 1g/L, peptone 7g/L, K ₂HPO₄ 0.5g/L,KH₂PO₄ 0.5g/L,MgSO₄·7H₂ O0.5 g/L, monosodium glutamate 1g/L, water, pH=7.5.

Rhodococcus fermentation medium: glucose 30g/L, yeast extract 7-8g/L, urea 10g/L,K₂HPO₄ 1.74g/L,KH₂PO₄ 0.866g/L,MgSO₄·7H₂O 1g/L, monosodium glutamate 1g/L, water, pH=7.5.

Other fusion expression recombinant vectors were constructed according to the method of example 3, and recombinant rhodococcus was constructed according to the method of example 5, followed by SDS-PAGE identification according to the method of this example.

The results are shown in FIG. 10. In fig. 10: strip 1: standard molecular weight proteins; strip 2: recombinant rhodococcus R.ruber BP4-mCherry broth supernatant; strip 3: recombinant rhodococcus R.ruber BP10-mCherry broth supernatant; strip 4: recombinant rhodococcus R.ruber BP12-mCherry broth supernatant; strip 5: recombinant rhodococcus R.ruber BP13-mCherry broth supernatant; strip 6: recombinant rhodococcus R.ruber BP14-mCherry broth supernatant; strip 7: recombinant rhodococcus R.ruber BP15-mCherry broth supernatant; strip 8: recombinant rhodococcus R.ruber BP16-mCherry broth supernatant; strip 9: recombinant rhodococcus R.ruber BP18-mCherry broth supernatant.

The embodiment of the application provides a series of rhodococcus-derived polypeptides which have the dual functions of mediating target protein secretion and enhancing protein expression level. After the encoding gene of the polypeptide and the target gene are fused and expressed in an operable way, the efficient secretory expression of the target protein is realized. The recombinant strain constructed by the application is used for secreting and expressing red fluorescent protein mCherry, and the extracellular fluorescent intensity of the induced expression for 48 hours can reach 7.6X10 ⁴. The polypeptide sequence fills the gap of the research of the rhodococcus secretion polypeptide library, and establishes an important secretion expression system of the industrial application strain rhodococcus.

The technical features of the above-described embodiments and examples may be combined in any suitable manner, and for brevity of description, all of the possible combinations of the technical features of the above-described embodiments and examples are not described, however, as long as there is no contradiction between the combinations of the technical features, they should be considered to be within the scope described in the present specification.

The above examples merely represent a few embodiments of the present application, which facilitate a specific and detailed understanding of the technical solutions of the present application, but are not to be construed as limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Further, it is understood that various changes and modifications of the present application may be made by those skilled in the art after reading the above teachings, and equivalents thereof are intended to fall within the scope of the present application. It should also be understood that, based on the technical solutions provided by the present application, those skilled in the art obtain technical solutions through logical analysis, reasoning or limited experiments, all of which are within the scope of protection of the appended claims. The scope of the patent is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted as illustrative of the contents of the claims.

Claims

1. A polypeptide having a length of 20aa to 50aa and an amino acid sequence as set forth in seq id no:

M（X）_n- RX-（X）_m（A）_p-（X）_q-AXA（XXA，AXX），

wherein,

M (X) _n -RX is the N-terminal charged region;

(X) _m（A）_p-（X）_q is an alanine-containing hydrophobic region;

AXA (XXA, AXX) is the N-terminal charged region;

The amino acid sequence of the polypeptide is shown in any one of SEQ ID NO.1, SEQ ID NO.3 to SEQ ID NO.5, SEQ ID NO.12 to SEQ ID NO.17, SEQ ID NO.19 to SEQ ID NO.43, SEQ ID NO.45 to SEQ ID NO.46 and SEQ ID NO. 76.

2. A nucleic acid molecule comprising a gene fragment of interest encoding a protein of interest and a polypeptide gene fragment encoding the polypeptide of claim 1, wherein the polypeptide gene fragment is linked to the 5' end of the gene fragment of interest; the target protein coded by the target gene fragment is endoglucanase or red fluorescent protein.

3. An expression vector comprising the nucleic acid molecule of claim 2.

4. A genetically engineered bacterium comprising the nucleic acid molecule of claim 2 or the expression vector of claim 3.

5. The genetically engineered bacterium of claim 4, comprising a bacterium.

6. The genetically engineered bacterium of claim 5, wherein the bacterium comprises rhodococcus, bacillus subtilis, bacillus licheniformis, corynebacterium glutamicum, or escherichia coli.

7. The genetically engineered bacterium of claim 6, wherein the rhodococcus comprises rhodococcus erythropolis or rhodococcus cloudus.

8. The method for constructing a genetically engineered bacterium according to any one of claims 4 to 7, comprising the step of introducing the nucleic acid molecule according to claim 2 into a host cell to construct a genetically engineered bacterium.

9. A method for producing a target protein, comprising culturing the genetically engineered bacterium of any one of claims 4 to 7; and isolating the protein of interest from the resulting culture.