CN117586356B - Polypeptides and their uses - Google Patents

Abstract

The present application relates to the fields of biotechnology and genetic engineering, and in particular to a polypeptide and its use. The polypeptide, a polypeptide, has a length of 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 integers of 0-10, and each occurrence of X is independently any amino acid; M(X) _n ‑RX is the N-terminal charged region; (X) _m （A） _p ‑(X) _q is a hydrophobic region containing alanine; AXA(XXA, AXX) is the N-terminal charged region. Fusion expression of the polypeptide at the N-terminus of the target protein can achieve secretory expression of the target protein in the host, and can also enhance the expression level of the target protein, that is, 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 their uses

Technical Field

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

Background technique

In the process of exogenous expression of target proteins, compared with intracellular expression, secretory expression has unique advantages and a wide range of application scenarios. For biocatalysis, some macromolecular substrates cannot cross the cell membrane. Only by secreting the enzyme outside the cell can the substrate molecules and enzymes be fully in contact, such as catalyzing the decomposition reaction of polymers such as lignin and cellulose. In addition, for heterologous protein production, the secretion strategy also has obvious advantages: first, secretion of target proteins can effectively avoid the aggregation of intracellular soluble proteins to form inclusion bodies; second, it is more suitable for expressing some proteins that are toxic to cells; third, because the bacteria are in a reduced state inside the cell, some proteins with disulfide bonds cannot be correctly folded inside the cell, and secretory expression can effectively solve this problem; finally, secretory proteins can effectively simplify the protein separation and purification steps and greatly reduce production costs. Therefore, it is crucial to improve the host's ability to secrete and express target proteins.

Summary of the invention

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

In the first aspect of the embodiments of the present application, a polypeptide is provided, 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),

in,

n, m, p, q are any integers from 0 to 10, and each occurrence of X is independently any amino acid;

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

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

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

In some embodiments of the present application, the amino acid sequence of the polypeptide is as shown in any one of SEQ ID NO.1 to SEQ ID NO.50, or has one or more amino acid mutations compared to the sequence shown in any one of SEQ ID NO.1 to SEQ ID NO.50 and the function of the polypeptide remains unchanged after the mutation, and the mutation includes insertion, deletion or substitution.

In a second aspect of the embodiments of the present application, a nucleic acid molecule is provided, which includes a polypeptide gene fragment encoding the polypeptide described in the first aspect.

In some embodiments of the present application, the nucleic acid molecule further includes a target gene fragment encoding a target protein and the polypeptide gene fragment is connected to the 5' end of the target gene fragment.

In some embodiments of the present application, the target gene fragment includes an enzyme.

In a third aspect of the embodiments of the present application, an expression vector is provided, which includes the nucleic acid molecule described in the second aspect.

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

In some embodiments of the present application, the genetically engineered bacteria include bacteria.

In some embodiments of the present application, the bacteria include Rhodococcus, Bacillus subtilis, Bacillus licheniformis, Corynebacterium glutamicum or Escherichia coli.

In some embodiments of the present application, the Rhodococcus includes Rhodococcus rubrum or Rhodococcus opacus.

In a fifth aspect of the embodiments of the present application, a method for constructing the genetically engineered bacteria described in the fourth aspect is provided, which comprises the step of introducing the nucleic acid molecule described in the second aspect into a host to construct the genetically engineered bacteria.

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

Compared with traditional technology, the implementation of this application has the following beneficial effects:

The embodiment of the present application provides a polypeptide, which is fused to the N-terminus of a target protein to achieve secretory expression of the target protein in a host and to enhance the expression level of the target protein, that is, the polypeptide has the dual functions of mediating the secretion of the target protein and enhancing the expression level of the target protein.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

Figure 1 is a schematic diagram of a polypeptide structure;

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

FIG3 is a schematic diagram of the construction of plasmid expression vectors with different polypeptide gene fragments;

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

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

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

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

FIG8 is a comparison of the secretion effects of BP6 and BP6 mutants;

FIG9 is a diagram showing the phenomenon of transparent "hydrolysis circle" around the recombinant strain obtained by connecting different polypeptide gene fragments to endoglucanase;

FIG. 10 is SDS-PAGE of the supernatant of the fermentation broth of recombinant Rhodococcus rubrum secreting the red fluorescent protein mCherry for 48 hours.

Detailed ways

The present invention will be further described in detail below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The purpose of providing these embodiments and examples is to make the understanding of the disclosure of the present invention more thorough and comprehensive. It should also be understood that the present invention can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various changes or modifications without violating the connotation of the present invention, and the equivalent form obtained also falls within the protection scope of the present application. In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the present invention. It should be understood that the present invention can be implemented without one or more of these details.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing implementation modes and embodiments and are not intended to limit the present invention.

the term

Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

The terms "and/or", "or/and", and "and/or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and/or", "or/and", and "and/or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and/or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and/or, B, and/or, C, and/or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

In the present invention, "plurality", "multiple", "multiple times", "multiple", etc., unless otherwise specified, refer to a number greater than or equal to 2. For example, "one or more" means one or greater than or equal to two.

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

Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of the present invention, solve the technical problem of the present invention, and achieve the expected technical effect of the present invention.

Herein, “preferred”, “better”, “more preferred” and “suitable” are merely used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute limitations on the scope of protection of the present invention.

In the present invention, “further”, “furthermore”, “particularly”, etc. are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present invention.

In the present invention, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel solutions of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

In the present invention, in the "first aspect", "second aspect", "third aspect", "fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

In the present invention, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum value and the maximum value) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the integer group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all sub-ranges included therein.

The temperature parameters in the present invention, if not specifically limited, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

In the present invention, %(w/w) and wt% both represent weight percentage, %(v/v) refers to volume percentage, and %(w/v) refers to mass volume percentage.

All documents mentioned in the present invention are cited as references in this application, just as each document is cited as a reference separately. Unless they conflict with the invention purpose and/or technical solution of the present application, the cited documents involved in the present invention are cited with all contents and all purposes. When the present invention involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present invention involves cited documents, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement the present invention. It should be understood that when the content of the citation conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

In the process of protein secretion, the N-terminal sequence of the target protein is particularly important. In order to achieve secretory expression of the protein, a polypeptide sequence with secretory function must be fused to the N-terminus of the sequence encoding the target protein. The latest research shows that the N-terminus of the protein also affects factors such as the biosynthesis, folding dynamics, and stability of the target protein. The embodiment of the present application expresses the polypeptide fusion at the N-terminus of the target protein, thereby achieving secretory expression of the target protein.

The first aspect of the present application

The present application example provides a polypeptide having a length of 20aa-50aa, and an amino acid sequence as shown below:

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

in,

n, m, p, q are any integers from 0 to 10, and each occurrence of X is independently any amino acid;

M(X) _n -RX is the charged region at the N-terminus (i.e., region 1);

(X) _m (A) _p - (X) _q is the hydrophobic region containing alanine (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 it can be AXA, XXA, or AXX.

According to the above amino acid sequence, it can be understood that region 1 is the N-terminal charged region, starting with methionine M and ending with RX (for example, RR). In region 2, (A) _p is between (X) _m and (X) _q . Region 3 is the C-terminal charged region, ending with three characteristic sequences: AXA or XXA, AXX.

Optionally, the amino acid sequence of the polypeptide is as shown in any one of SEQ ID NO.1 to SEQ ID NO.50, or has one or more amino acid mutations compared to the sequence shown in any one of SEQ ID NO.1 to SEQ ID NO.50, and the function of the polypeptide after the mutation remains unchanged, and the mutation includes insertion, deletion or substitution. Optionally, the number of amino acid mutations is 1 to 5 (for example, 1, 2, 3, 4, 5). Optionally, the substitution includes conservative substitution, especially conservative substitution 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.

Optionally, 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 BP51.

The polypeptides provided in the examples of the present application may exist alone, and polypeptides of different sequences may also be combined into a polypeptide library.

In the art, the above-mentioned types of mutations usually do not change the function of the polypeptide, especially when replacing it with an amino acid with similar properties, that is, conservative substitution.

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

Table A

In the second aspect of the embodiment of the present application

An embodiment of the present application provides a nucleic acid molecule, which includes a polypeptide gene fragment encoding the polypeptide described in the first aspect.

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

The examples of the present application do not specifically limit the types of target proteins encoded by the target gene fragments, including but not limited to enzymes, such as single-subunit enzymes or polymerases composed of the same subunits, which may be endoglucanase, exoglucanase, β-glucosidase, red fluorescent protein, lipase, nattokinase, etc., or mutants of these enzymes. The amino acid sequence of the target protein may be as shown in any of SEQ ID NO.51-SEQ ID NO.55, or may be a sequence having a high homology (e.g., not less than 90%) with the sequence shown in any of SEQ ID NO.51-SEQ ID NO.55.

SEQ ID NO.51 (885aa):

MKKRLVKKVAMLIAIVLVLSSSIGQAFALVGAGDLIRNHTFDNRVGLPWHVVESYPAKASFEITSDGKYKITAQKIGEAGKGERWDIQFRHRGLALQQGHTYTVKFTVTASRACKIYPKIGDQGDPYDEYWNMNQQWNFLELQANTPKTVTQTFTQTKGDKKNVEFAFHLAPDKTTSEAQNPASFQPITYTFDEIYIQDPQFAGYTEDPPEPTNVVRLNQV GFYPNADKIATVATSSTTPINWQLVNSTGAAVLTGKSTVKGADRASGDNVHIIDFSSYTTPGTDYKIVTDVSVTKAGDNESMKFNIGDDLFTQMKYDSMKYFYHNRSAIPIQMPYCDQSQWARPAGHTTDILAPDPTKDYKANYTLDVTGGWYDAGDHGKYVVNGGIATWTVMNAYERALHMGGDTSVAPFKDGSLNIPESGNGYPDILDEARYNMKTLLNM QVPAGNELAGMAAHHKAHDERWTALAVRPDQDTMKRWLQPPSTAATLNLAAIAAQSSRLWKQFDSAFATKCLTAAETAWDAAVAHPEIYATMEQGAGGGAYGDNYVLDDFYWAACELYATTGSDKYLNYIKSSKHYLEMPTELTGGENTGITGAFDWGCTAGMGTITLALVPTKLPAADVATAKANIQAAADKFISISKAQGYGVPLEEKVISSPFDASVVK GFQWGSNSFVINEAIVMSYAYEFSDVNGTKNNKYINGALTAMDYLLGRNPNIQSYITGYGDNPLENPHHRFWAYQADNTFPKPPPGCLSGGPNSGLQDPWVKGSGWQPGERPAEKCFMDNIESWSTNEITINWNAPLVWISAYLDEKGPEIGGSVTPPTNLGDVNGDGNKDALDFAALKKALLSQDTSTINVANADINKDGSIDAVDFALLKSFLLGKITL.

SEQ ID NO.52 (475aa):

MKKTTAFLLCFLMIFTALLPMQNANAYDASLIPNLQIPQKNIPNNDGMNFVKGLRLGWNLGNTFDAFNGTNITNELDYETSWSGIKTTKQMIDAIKQKGFNTVRIPVSWHPHVSGSDYKISDVWMNRVQEVVNYCIDNKMYVILNTHHDVDKVKGYFPSSQYMASSKKYITSVWAQIAARFANYDEHLIFEGMNEPRLVGHANEWWPELTNSDVVDSINCINQLNQDFVNTVRATGGK NASRYLMCPGYVASPDGATNDYFRMPNDISGNNNKIIVSVHAYCPWNFAGLAMADGGTNAWNINDSKDQSEVTWFMDNIYNKYTSRGIPVIIGECGAVDKNNLKTRVEYMSYYVAQAKARGILCILWDNNNFSGTGELFGFFDRRSCQFKFPEIIDGMVKYAFEAKTDPDPVIVYGDYNNDGNVDALDFAGLKKYIMAADHAYVKNLDVNLDNEVNAFDLAILKKYLLGMVSKLPSN.

SEQ ID NO.53 (711aa):

MQYDQIDKKIDELLSMMTLEEKAGMCHGAGLFRTAGVPRLGIPPLVFSDGPMGIRNEFADDNWNTVGGNTDFVTYLPANTALAATFNRTLAESLGEVLGCEARGRGKDVILAPGVNIIRTPLCGRNYEYFSEDPILTAELAASFIKGVQRFDVAACVKHFAANNQETERLAVSAEVDELTLRELYFPAFEASVRAGVLTVMTAYNRLNGTFCSHSRQLITEILREEWGFNGVVVSDWGAVHDTESPAIAGLDIEMNVTSNFNEYFFAKPLINAIKDGKIPERMLDDKVRRILRLMFRLNMFSKDRKRGGFNLPQHQQAVLDAAKESFVLLKNDREVLPLNADGIKTVAVIGSNADK KHSSGGDSAAVKALYEVTPLSGIVMRLASGAKVTYYPGCPDETHYKEEFHIPSNADEKTRADIEEKARAADEDYRKIQMRLEDEAIQAAKTADAVIFIGGNGHEQESEGRDRPDMALPYEQDKLLSRVLDANPNTVVVIISGSPVNMSGWIDKAPTVMQGFFSGMHGGTALAAVLFGDENPSGHLPFTIPLKEEETGASALGEYPGGETVCYSEGLFVGYRYHDAFNIPPLFPFGYGLSYTTFSLANESFRRLPCVGTEYEIHVDITNSGNRPGAQSVQLYVEPEKKDGSPIRTLKGFEKTYLNPDETKTITFKLDERTFSEFRPHEGWVFVPGNYTIHIGTSSRELPIAIALAL.

SEQ ID NO.54 (236aa)

MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK.

SEQ ID NO.55 (270aa):

MSINGGIRAATSQEINELTYYTTLSANSYCRTVIPGATWDCIHCDATEDLKIIKTWSTLIYDTNAMVARGDSEKTIYIVFRGSSSIRNWIADLTFVPVSYPPVSGTKVHKGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGATVLLCALDLYQREEGLSSSNLFLYTQGQPRVGDPAFANYVVSTGIPYRRTVNERDIVPHLPPAAFGFLHAGEEYWITDNSPETVQVCTSDLETSDCSNSIVPFTSVLDHLSYFGINTGLCT.

The third aspect of the embodiment of the present application

An embodiment of the present application provides an expression vector, which includes the nucleic acid molecule described in the second aspect.

The fourth aspect of the embodiment of the present application

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

The embodiments of the present application do not specifically limit the types of genetically engineered bacteria, for example, bacteria can be selected. The present application does not specifically limit the types of bacteria, for example, it can be Rhodococcus (such as Rhodococcus ruber, Rhodococcus opacus), Bacillus subtilis, Bacillus licheniformis, Corynebacterium glutamicum, Escherichia coli, etc. Among them, Rhodococcus is an aerobic Gram-positive bacterium and a non-model strain. Due to its high tolerance to organic solvents and rich nitrile and aromatic compound metabolic enzyme system, Rhodococcus has a wide range of applications in the fields of biocatalysis, environmental remediation, biosynthesis, and wood degradation and utilization. For example: in the field of biocatalysis, Rhodococcus has been successfully used in the biological production of compounds such as acrylamide and acrylic acid; in the field of biosynthesis, Rhodococcus can synthesize many high value-added products such as biosurfactants, carotenoids, antibacterial agents, triglycerides and polyhydroxyalkanoates. In addition, Rhodococcus is also an efficient protein expression host. For example, in Rhodococcus rhodochrous, nitrile hydratase can account for about 45% of the total soluble protein. The above application areas all achieve biocatalysis, biosynthesis and other purposes by expressing the target protein in Rhodococcus cells. In view of the many characteristics of Rhodococcus and its important potential application value, improving the secretory expression ability of Rhodococcus for target proteins plays an important role in broadening the application field of Rhodococcus and developing Rhodococcus catalysis and synthesis platforms.

The genetically engineered bacteria of the embodiments of the present application may contain the above-mentioned vector, or may have the above-mentioned nucleic acid molecule integrated into the genome.

A fifth aspect of the embodiments of the present application

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

The construction method of the embodiment of the present application includes but is not limited to the following steps: connecting the above nucleic acid molecule into the Escherichia coli-Nocardia/Rhodococcus shuttle plasmid, constructing a recombinant plasmid vector, and transforming it into a host cell. Further, the shuttle plasmid can be one of pNV18, pNV18.1, pNV19, pRC, pφC31 plasmids or their derivative plasmids.

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

The inventors analyzed and identified the secretory protein group of Rhodococcus, and analyzed and identified the secretory protein sequences from Rhodococcus recorded in the database, and after multiple screening, 50 polypeptides that mediate the secretory expression of the target protein were obtained. The embodiments of the present invention will be described in detail below in conjunction with the examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. For experimental methods in the following examples where specific conditions are not specified, please refer to the instructions given in the present invention first, and you can also follow the experimental manuals or conventional conditions in the field, or the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

In the following specific examples, the structure of the polypeptide involved is shown in FIG1 , and the schematic diagram of the construction of the recombinant vector using pNV18.1-Pa2 (see FIG2 ) is shown in FIG3 .

Example 1. Construction of the recombinant vector pNV-18.1-Pa2-BP-eg carrying an exogenous endoglucanase gene

1. The endoglucanase gene from Clostridium cellulolyticum was selected and codon optimized for the Rhodococcus rubrum host. The nucleotide sequence of the gene after optimization is shown in SEQ ID NO.56. The gene synthesis work was entrusted to Beijing Liuhe BGI Technology Co., Ltd. SEQ ID NO.56 is as follows:

ATGAAGAAGACCACCGCCTTCCTGCTGTGCTTCCTGATGATCTTCACCGCCCTGCTGCCGATGCAGAACGCCAATGCCTATGATGCCTCGCTGATTCCGAATCTGCAGATCCCGCAAAAGAACATCCCGAACAACGACGGCATGAACTTCGTCAAGGGCCTGCGCTTAGGCTGGAACCTGGGCAATACCTTTGACGCCTTTAACGGCACCAACATCACCAACGAGCTGGACTACGAGACCTCGTGGTCGGGCATTAAGACCACCAAACAGATGATCGACGCCATCAAGCAGAAGGGCTTCAACACCGTCCGCATCCCGGTCTCGTGGCATCCGCATGTCTCGGGCTCGGATTATAAG ATTTCGGACGTCTGGATGAACCGCGTCCAGGAGGTCGTTAATTACTGCATCGACAACAAGATGTACGTCATCCTGAACACCCACCACGACGTCGACAAGGTCAAGGGCTATTTCCCGTCGTCGCAGTACATGGCCTCGTCGAAAAAGTACATCACCTCGGTCTGGGCCCAGATCGCCGCACGTTTTGCCAATTATGATGAACATCTGATCTTCGAGGGCATGAACGAGCCGCGCCTGGTCGGCCATGCCAATGAATGGTGGCCGGAACTGACCAATTCGGACGTCGTCGACTCGATTAACTGCATCAACCAGCTGAACCAGGACTTCGTCAACACCGTCCGCGCCACCGGCGGTAAA AATGCCTCGCGCTATCTGATGTGCCCGGGCTATGTTGCCTCGCCGGATGGCGCCACCAATGATTATTTCCGCATGCCGAACGACATCTCGGGCAACAACAACAAGATCATCGTCTCGGTCCACGCCTACTGCCCGTGGAATTTTGCCGGCCTGGCCATGGCCGATGGCGGCACCAACGCCTGGAACATTAACGATTCGAAGGACCAGTCGGAGGTCACCTGGTTCATGGACAACATCTACAACAAGTACACCTCGCGCGGCATCCCGGTCATTATTGGCGAATGCGGCGCCGTCGATAAAAATAATCTGAAGACCCGCGTCGAGTACATGTCGTACTACGTCGCCCAGGCCAAGGCC CGCGGTATACTGTGTATTCTGTGGGACAATAACAACTTCTCGGGCACCGGCGAGCTGTTCGGCTTTTTTGATCGCCGCTCGTGTCAGTTCAAGTTCCCGGAGATAATCGACGGCATGGTCAAGTACGCCTTCGAGGCCAAAACCGACCCGGACCCAGTTATTGTTTATGGCGATTACAATAACGACGGCAACGTCGACGCCCTGGACTTCGCAGGCTTGAAAAAGTATATCATGGCCGCCGACCACGCCTACGTCAAGAATCTGGATGTCAACCTGGACAACGAGGTCAACGCCTTCGACCTGGCCATTCTGAAGAAGTACCTGCTGGGCATGGTCTCGAAGCTGCCGTCGAATTGA.

2. Using eg6-F/eg6-R, eg7-F/eg7-R, and eg8-F/eg8-R as primers, and the gene sequence synthesized in the above steps as a template, PCR reaction was performed to amplify the endoglucanase gene eg, and then recovered and purified. Using BP6-F/BP6-R, BP7-F/BP7-R, and BP8-F/BP8-R as primers, and using the genomic DNA of Rhodococcus as a template, the genes of polypeptides BP6 (SEQ ID NO.6), BP7 (SEQ ID NO.7), and BP8 (SEQ ID NO.8) were amplified, and then recovered and purified. The plasmid backbone pNV18.1-Pa2 (as shown in Figure 2) was double-digested with restriction endonucleases XbaI and KpnI, and the linearized plasmid was recovered and purified. The Gibson Assembly kit (Clonesmarter brand, purchased from Sino-US Taihe Biotechnology (Beijing) Co., Ltd.) was used to construct three connection systems, namely: linearized vector + BP6 + eg; linearized vector + BP7 + eg; linearized vector + BP8 + eg.

3. The ligation product of step 2 was transformed into Escherichia coli trans10 competent cells (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) by chemical transformation. The specific operation steps are as follows: 10 μL of the ligation system was added to 100 μL of competent cells and placed on ice for 30 minutes. After heat shock at 42°C for 45 seconds, it was placed on ice for 2 minutes. Then 900 μL of resistance-free SOC medium was added and the cells were placed in a shaker for 60 minutes for recovery. The recovery conditions were: 200 rpm, 37°C. After the recovery, 200 μL of bacterial solution was spread on a solid plate of LB medium containing 50 μg/mL kanamycin, positive clones were selected, and PCR verification and sequencing verification were performed to obtain the recombinant vectors pNV18.1-Pa2-BP6-eg (as shown in Figure 4), pNV18.1-Pa2-BP7-eg (as shown in Figure 5), and pNV18.1-Pa2-BP8-eg (as shown in Figure 6).

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

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

The primers were synthesized by Suzhou Anshengda Biotechnology Co., Ltd. and diluted to 10 μM with sterile water. PCR amplification used the high-fidelity DNA polymerase Phanta Max Master 2×mix from Nanjing Novozyme.

The sequences of the primers (5'-3') 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): AAGCCGCCCCCGTATGATGCCTCGCTG;

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

PCR reaction:

The PCR reaction system was: template 1 μL, upstream primer 2 μL, downstream primer 2 μL, Phanta Max MasterMix 25 μL, sterile water 20 μL, total volume 50 μL;

PCR reaction conditions were: 95°C for 3 min; 95°C for 30 s, 55°C for 15 s, 72°C for 2 min, 35 cycles; 72°C for 5 min; 4°C forever.

Example 2: Construction of recombinant plasmid pNV-18.1-Pa2-BP6-mut-eg carrying BP6 point mutant

1. The last amino acid of BP6 polypeptide was mutated to T38A. The construction process is as follows:

The recombinant plasmid pNV18.1-Pa2-BP6-eg constructed in Example 1 was used as a template, and BP6-mut-F/BP6-mut-R was used as primers to perform PCR to linearize the plasmid, and the PCR product was recovered and purified. The linearized vector was connected using a Gibson Assembly kit (Clonesmarter brand, purchased from Sino-US Taihe Biotechnology (Beijing) Co., Ltd.).

2. The ligation product was transformed into Escherichia coli trans10 competent cells (purchased from Beijing Quanshijin Biotechnology Co., Ltd., and the specific operation steps are shown in Example 1) by chemical transformation. The recombinant vector pNV18.1-Pa2-BP6-mut-eg was obtained.

The PCR reaction system was as follows: template 1 μL (1 ng/μL), upstream primer 2 μL, downstream primer 2 μL, Phanta MaxMaster Mix 25 μL, sterile water 20 μL, total volume 50 μL;

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

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

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

Example 3: Construction of the recombinant vector pNV-18.1-Pa2-BP10-mCherry carrying the red fluorescent protein gene mcherry

1. The red fluorescent protein mCherry gene was selected and codon optimized for the red coccus host. The nucleotide sequence of the gene after optimization is shown in SEQ ID NO.57. The gene synthesis work was entrusted to Beijing Liuhe BGI Technology Co., Ltd. SEQ ID NO.57 is as follows:

ATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCA GGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTGA.

2. Using mCherry-F/mCherry-R as primers and the gene sequence synthesized in the above steps as a template, PCR amplify the mCherry gene fragment and recover and purify it. Using BP10-F/BP10-R as primers and the genomic DNA of Rhodococcus rubrum as a template, amplify the gene sequence of the polypeptide sequence BP10, and recover and purify it. Use restriction endonucleases XbaI and KpnI to double-digest the plasmid backbone pNV18.1-Pa2, and recover and purify the linearized plasmid. Use the Gibson Assembly kit (Clonesmarter brand, purchased from Sino-US Taihe Biotechnology (Beijing) Co., Ltd.) for connection, and the connection system is: linearized vector + BP10 + mCherry.

3. The ligation product was transformed into Escherichia coli trans10 competent cells (purchased from Beijing Quanshijin Biotechnology Co., Ltd., and the specific operation steps are shown in Example 1) by chemical transformation. The recombinant vector pNV18.1-Pa2-BP10-mCherry was obtained (as shown in Figure 7).

The culture medium formula is shown in Example 1.

The primer sequences (5'-3') are 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 bacteria that secrete and express exogenous endoglucanase

The recombinant plasmids constructed in Example 1 and Example 2 were respectively transformed into Rhodococcus ruber THdAdN by electroporation (the construction method is detailed in Chinese invention patent application CN105420154A), and recombinant Rhodococcus ruber BP6-EG, R. ruber BP6-mut-EG, R. ruber BP7-EG and R. ruber BP8-EG were obtained accordingly. The specific operation method is as follows: add 10 μL of the above recombinant plasmid to 80 μL R. ruber THdAdN competent cells, mix well, ice bath for 10 minutes, and transfer to a 1 mm electroporation cup; adjust the voltage of the electroporator to 1.25 kV, install the electroporation cup into the electroporator, and press the electroporation button; immediately add 900 μL LBHIS culture medium to the electroporation cup after the electroporation, resuspend the cells, and transfer to a 1.5 mL centrifuge tube; 28°C, 200 rpm recovery for 2.5 hours; after the recovery, high-speed centrifugation at 13000 rpm and remove 800 μL of supernatant, resuspend the centrifuged cells, and spread them on Rhodococcus solid culture medium (kanamycin 25 μg/mL), place them in an incubator at 28°C and culture them upside down for 48-60 hours, and select positive clones to obtain recombinant Rhodococcus that can secrete and express exogenous endoglucanase.

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

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

Example 5: Construction of recombinant Rhodococcus bacteria that secrete and express red fluorescent protein

The recombinant plasmid constructed in Example 3 was transformed into R. ruber THdAdN by electroporation (the construction method is detailed in Chinese invention patent application CN105420154 A) to obtain recombinant R. ruber BP10-mCherry. The specific operation method is the same as that in Example 4. Positive clones were selected to obtain recombinant R. ruber that can secrete red fluorescent protein mCherry.

Example 6: Determination of the activity of recombinant Rhodococcus extracellular endoglucanase

The Congo red hydrolysis circle method was used to determine the extracellular endoglucanase activity of the recombinant Rhodococcus. The specific operation method is as follows: the recombinant Rhodococcus constructed in Example 4 was streaked on the solid culture medium of Rhodococcus, and a single colony was picked and inoculated on the solid fermentation medium of Rhodococcus containing sodium carboxymethyl cellulose (CMC-Na, 1%), and placed in a 28°C incubator for 48 hours, and then stained with 20mL of Congo red dye for 30 minutes, and then decolorized with 1M NaCl solution for 30 minutes. The diameter of the hydrolysis circle was measured in two perpendicular directions to characterize the activity of the extracellular endoglucanase.

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

Other fusion expression recombinant vectors were constructed by referring to the method of Example 1, and recombinant Rhodococcus was constructed by referring to the method of Example 2, and endoglucanase activity was tested. The results are shown in Table 1, Table 2, Figure 8 and Figure 9.

Table 1

Table 2

The amino acid sequence of BP51 (BP6-mut, T38A) is: MQTGTSRGMKRLAGGAALAAAAAATVAVTMPATASAAA (SEQ ID NO. 76).

Example 7: Denaturing polyacrylamide gel electrophoresis of recombinant Rhodococcus extracellular red fluorescent protein

The recombinant Rhodococcus constructed in Example 5 was activated on a plate, and a single colony was picked and inoculated into a Rhodococcus seed culture medium, and cultured at 28°C 200rpm for 48-60h; 10% of the inoculum was inoculated into a Rhodococcus fermentation medium, and fermented at 28°C 200rpm for 48 hours. After the fermentation was completed, a certain volume of fermentation liquid was taken, centrifuged at 13000rpm for 10 minutes to remove the bacteria, and the fermentation liquid supernatant was collected for SDS-PAGE identification.

Rhodococcus seed culture medium: glucose 20 g/L, yeast extract 1 g/L, peptone 7 g/L, K ₂ HPO ₄ 0.5 g/L, KH ₂ PO ₄ 0.5 g/L, MgSO ₄ ·7H ₂ O 0.5 g/L, MSG 1 g/L, water, pH = 7.5.

Rhodococcus fermentation medium: glucose 30 g/L, yeast extract 7-8 g/L, urea 10 g/L, K ₂ HPO ₄ 1.74 g/L, KH ₂ PO ₄ 0.866 g/L, MgSO ₄ ·7H ₂ O 1 g/L, MSG 1 g/L, water, pH=7.5.

Other fusion expression recombinant vectors were constructed by referring to the method of Example 3, and recombinant Rhodococcus was constructed by referring to the method of Example 5, and then SDS-PAGE identification was performed by referring to the method of this example.

The results are shown in Figure 10. In Figure 10: Band 1: standard molecular weight protein; Band 2: recombinant Rhodococcus R. ruber BP4-mCherry fermentation broth supernatant; Band 3: recombinant Rhodococcus R. ruber BP10-mCherry fermentation broth supernatant; Band 4: recombinant Rhodococcus R. ruber BP12-mCherry fermentation broth supernatant; Band 5: recombinant Rhodococcus R. ruber BP13-mCherry fermentation broth supernatant; Band 6: recombinant Rhodococcus R. ruber BP14-mCherry fermentation broth supernatant; Band 7: recombinant Rhodococcus R. ruber BP15-mCherry fermentation broth supernatant; Band 8: recombinant Rhodococcus R. ruber BP16-mCherry fermentation broth supernatant; Band 9: recombinant Rhodococcus R. ruber BP18-mCherry fermentation broth supernatant.

The embodiments of the present application provide a series of polypeptides derived from Rhodococcus, which have the dual functions of mediating the secretion of target proteins and enhancing the protein expression level. After the coding gene of the polypeptide and the target gene are operably fused and expressed, efficient secretory expression of the target protein is achieved. The recombinant strain constructed by the present invention secretes and expresses the red fluorescent protein mCherry, and the extracellular fluorescence intensity can reach up to 7.6×10 ⁴ after induced expression for 48 hours. The polypeptide sequence provided in this application fills the gap in the research of the secretory polypeptide library of Rhodococcus, and establishes a secretory expression system of the important industrial application strain Rhodococcus.

The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

The above-described embodiments only express several implementation methods of the present invention, which is convenient for understanding the technical solution of the present invention in detail, but it cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present invention, several variations and improvements can be made, which all belong to the protection scope of the present invention. In addition, it should be understood that after reading the above-mentioned teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and the equivalent forms obtained also fall within the protection scope of this application. It should also be understood that the technical solutions obtained by those skilled in the art on the basis of the technical solution provided by the present invention through logical analysis, reasoning or limited experiments are all within the protection scope of the claims attached to the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the content of the attached claims, and the description and drawings can be used to explain the content of the claims.

Claims (9)

1. A polypeptide with a length of 20aa-50aa and an amino acid sequence as shown below: M(X) _n -RX-(X) _m (A) _p- (X) _q -AXA(XXA,AXX), in, n, m, p, q are any integers from 0 to 10, and each occurrence of X is independently any amino acid; M(X) _n -RX is the N-terminal charged region; (X) _m (A) _p - (X) _q is the hydrophobic region containing alanine; 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 target gene fragment encoding a target protein and a polypeptide gene fragment encoding the polypeptide according to claim 1, wherein the polypeptide gene fragment is connected to the 5' end of the target gene fragment; the target protein encoded 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. The genetically engineered bacteria according to claim 4 , comprising bacteria. 6. The genetically engineered bacteria according to claim 5, wherein the bacteria include Rhodococcus, Bacillus subtilis, Bacillus licheniformis, Corynebacterium glutamicum or Escherichia coli. 7. The genetically engineered bacteria according to claim 6, wherein the Rhodococcus includes Rhodococcus rubrum or Rhodococcus opacus. 8. A 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 to construct the genetically engineered bacterium. 9. A method for producing a target protein, comprising culturing the genetically engineered bacterium according to any one of claims 4 to 7; and isolating the target protein from the obtained culture.