Novel LuxI protein derived from deep sea cold spring as well as coding gene and application of novel LuxI protein

Abstract

The invention provides a novel LuxI protein from a deep sea cold spring as well as a coding gene and application thereof. The amino acid sequence of the novel LuxI protein is as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. The novel LuxI protein from the deep sea cold spring extreme environment is obtained through a large amount of complex bioinformatics analysis and research, and efficient heterologous expression of the novel LuxI protein is successfully achieved. Furthermore, a biosensing strain experiment verifies that the obtained novel LuxI protein can specifically catalyze synthesis and secrete acyl homoserine lactone (AHLs) molecules, and the chromogenic reaction signal of the biosensing strain is stable and repeatable.

Description

Technical Field

[0001] This invention belongs to the field of microbiology technology, especially microbial quorum sensing, and specifically relates to a novel LuxI protein from deep-sea cold seeps, its encoding gene, and its applications. Background Technology

[0002] Quorum sensing is a complex intercellular communication mechanism mediated by autoinducers (AIs), often referred to as the "language" of microorganisms. It coordinates collective behaviors such as biofilm formation, cellular capabilities, movement, horizontal gene transfer, and symbiotic relationships, and is widely found in both Gram-negative and Gram-positive bacteria. The most typical quorum sensing system is mediated by acylhomoserine lactones (AHLs), typically relying on the LuxI-LuxR circuit to regulate intercellular cooperation and communication. LuxI proteins are autoinducer synthases responsible for the synthesis of AHL signaling molecules; LuxR is the corresponding receptor protein that senses and responds to AHLs. Traditional methods for discovering and validating QS systems often rely on culturable strains and complex chemical identification techniques, such as gene cloning and sequencing, validation through mutant construction and functional screening. These methods have drawbacks, such as low recognition efficiency and high cost. Especially in extreme environments, low-abundance signaling molecules are difficult to capture, and most microorganisms are unculturable, limiting the acquisition and application of novel QS elements and hindering large-scale production.

[0003] Existing research on LuxI, a quorum sensing protein, mainly focuses on a few model strains, resulting in limited source diversity and sequence diversity. This leads to insufficient understanding of the overall evolutionary relationships and functional characteristics of LuxI proteins. Existing studies on LuxI proteins often suffer from the following limitations: First, the catalytic efficiency and substrate specificity of LuxI proteins from different sources vary significantly, restricting their application in synthetic biology and novel stress resistance strategies. Second, some LuxI proteins exhibit low expression efficiency in commonly used heterologous hosts (such as *E. coli*), making it difficult to obtain sufficient quantities of functional proteins for experimental validation or application development. Third, existing LuxI databases have limited coverage, making it difficult to systematically mine potential novel LuxI proteins from complex or extreme environments.

[0004] Therefore, there is a need in this field to discover more types of LuxI proteins, especially novel LuxI proteins discovered from complex or extreme environments (such as deep-sea cold seeps). Summary of the Invention

[0005] Based on this, the purpose of the present invention is to provide a novel LuxI protein from deep-sea cold seeps, its encoding gene and applications. The novel LuxI protein is isolated from deep-sea cold seeps, can be efficiently expressed in a heterologous host, and can catalyze the synthesis of acylhomoserine lactones.

[0006] To achieve the above objectives, the present invention adopts the following technical solution.

[0007] A first aspect of the present invention is to provide an isolated protein having an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0008] A second aspect of the present invention is to provide an isolated polynucleotide encoding the protein as described in claim 1.

[0009] In some embodiments, the nucleotide sequence of the polynucleotide encoding the protein with the amino acid sequence shown in SEQ ID NO: 1 is shown in SEQ ID NO: 6 or SEQ ID NO: 11; The nucleotide sequence of the polynucleotide encoding the protein with the amino acid sequence shown in SEQ ID NO: 2 is shown in SEQ ID NO: 7 or SEQ ID NO: 12; The nucleotide sequence of the polynucleotide encoding the protein with the amino acid sequence shown in SEQ ID NO: 3 is shown in SEQ ID NO: 8 or SEQ ID NO: 13; The nucleotide sequences of the polynucleotides encoding the protein with the amino acid sequence shown in SEQ ID NO: 4 are shown in SEQ ID NO: 9 or SEQ ID NO: 14.

[0010] A third aspect of the present invention is to provide a recombinant expression vector having inserted polynucleotides as described above.

[0011] In some embodiments, the backbone vector of the recombinant expression vector is pET-28(a).

[0012] A fourth aspect of the present invention is to provide a recombinant engineered bacterium, the recombinant engineered bacterium comprising the protein, the polynucleotide, or the recombinant expression vector as described above.

[0013] In some embodiments, the host bacterium of the recombinant engineered bacteria is Escherichia coli, preferably Escherichia coli BL21(DE3).

[0014] A fifth aspect of the invention is the use of the protein, polynucleotide, recombinant expression vector, and / or recombinant engineered bacteria as described above in the preparation of acylhomoserine lactones.

[0015] A sixth aspect of the present invention is to provide a method for preparing acylhomoserine lactone, comprising the steps of: using the protein described above as a synthase to catalyze the synthesis of acylhomoserine lactone.

[0016] In some embodiments, the method includes the following steps: (1) culturing the recombinant engineered bacteria as described above; (2) adding an inducer to the culture system and continuing to culture; (3) collecting the bacterial cells, breaking them, and separating them to obtain the desired result.

[0017] In some embodiments, the inducer is isopropyl-β-D-thiogalactopyranoside.

[0018] In some embodiments, the concentration of isopropyl-β-D-thiogalactopyranoside in the culture system is 0.1 mM to 0.3 mM.

[0019] In some embodiments, the crushing method is ultrasonic crushing.

[0020] In some embodiments, the culture temperature in step (1) is 20±1°C.

[0021] In some embodiments, in step (2), when the OD of the culture system 600 When the concentration is 0.4-0.6, the inducing agent is added.

[0022] In some embodiments, the culture time in step (2) is 16h~20h and the temperature is 16±1°C.

[0023] Compared with the prior art, the present invention has the following beneficial effects.

[0024] This invention, through extensive bioinformatics analysis and research, obtained a novel LuxI protein derived from the extreme environment of deep-sea cold seeps. By predicting its structure, removing the signal peptide coding region from the corresponding coding gene sequence, and optimizing the codons, the efficient heterologous expression of the novel LuxI protein was successfully achieved. Furthermore, the invention's novel LuxI protein was verified using biosensor strains to specifically catalyze the synthesis and secretion of acylhomoserine lactones (AHLs), and the colorimetric reaction signal of the biosensor strains was stable and reproducible.

[0025] The novel LuxI protein described in this invention originates from a deep-sea cold seep environment. luxI The fact that this gene can still fold correctly and maintain its catalytic function in a heterologous host indicates that the protein has strong environmental adaptability and structural stability. This characteristic opens up possibilities for its signal synthesis and quorum sensing regulation under extreme environments, expands its potential for applications in industrial and environmental microbiology, and provides a new research subject for the overall evolutionary relationships and functional characteristics of LuxI-type proteins. Attached Figure Description

[0026] Figure 1The diagram shows the structure of the recombinant plasmid vector containing the optimized coding gene with the >HM_SQ_cobin_128_LuxI codon.

[0027] Figure 2 This is a structural diagram of a recombinant plasmid vector containing the optimized coding gene with the codon >P_20_sbin.39_LuxI inserted.

[0028] Figure 3 This is a structural diagram of a recombinant plasmid vector containing the optimized coding gene with the codon >X_3_6_sbin.12_LuxI inserted.

[0029] Figure 4 This is a structural diagram of a recombinant plasmid vector containing the optimized coding gene with the codon >X_6_9_sbin.73_LuxI inserted.

[0030] Figure 5 This is a structural diagram of the recombinant plasmid vector containing the optimized coding gene with the codon >XST_SQ83_0.10_sbin_20_LuxI.

[0031] Figure 6 For carrying luxI Genetic E. coli SDS-PAGE gel image of BL21 (DE3) lysis buffer.

[0032] Figure 7 For carrying luxI Genetic E. coli Figure showing the results of the indicator bacteria pigment experiment for BL21 (DE3).

[0033] Figure 8 Structural prediction and alignment of four LuxI protein sequences. Detailed Implementation

[0034] Experimental methods in the following embodiments of the present invention, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. All commonly used chemical reagents used in the embodiments are commercially available products.

[0035] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0036] The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device.

[0037] The term "and / or" as used in this invention describes the relationship between related objects, indicating that there can be three relationships. For example, A and / or B can represent three situations: A exists alone, A and B exist simultaneously, and B exists alone.

[0038] Some embodiments of the present invention relate to an isolated protein (a novel LuxI protein) having an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0039] This invention, through extensive and complex bioinformatics analysis and research, obtained the amino acid sequence of a novel LuxI protein derived from the extreme environment of deep-sea cold seeps. By predicting its signal peptide, removing the signal peptide coding region from the corresponding coding gene sequence, and optimizing the codons, the efficient heterologous expression of the novel LuxI protein was successfully achieved. Furthermore, experiments using biosensor strains verified that the obtained novel LuxI protein can specifically catalyze the synthesis and secretion of acylhomoserine lactones (AHLs), and the colorimetric reaction signal of the biosensor strains was stable and reproducible.

[0040] The novel LuxI protein described in this invention originates from a deep-sea cold seep environment. luxI The fact that this gene can still fold correctly and maintain its catalytic function in a heterologous host indicates that the protein has strong environmental adaptability and structural stability. This characteristic opens up possibilities for its signal synthesis and quorum sensing regulation under extreme environments, expands its potential for applications in industrial and environmental microbiology, and provides a new research subject for the overall evolutionary relationships and functional characteristics of LuxI-type proteins.

[0041] This invention presents a novel LuxI protein capable of efficiently synthesizing quorum sensing signaling molecules (AHLs), thus demonstrating broad potential in various application scenarios. Firstly, in synthetic biology, this type of LuxI can serve as a core component of quorum sensing regulatory circuits, enabling precise regulation of intercellular information transmission and quorum behavior, providing a powerful tool for constructing artificial synthetic communities, dynamically regulating metabolic pathways, and realizing intelligent cell factories. Secondly, AHLs not only play a role in signal transduction among bacteria but can also be sensed by eukaryotes (such as plants and animals), thereby participating in cross-species communication. For example, in plants, AHLs can regulate growth and development, induce defense responses, enhance disease resistance, and improve tolerance to abiotic stresses such as drought and salt stress; in animal cells, AHLs may exert immunomodulatory effects by influencing the immune system and inflammatory pathways. Furthermore, AHLs also show promise in microbial community metabolism and environmental management. AHLs can regulate the formation and function of biofilms, thereby improving the efficiency of microbial synergy in key metabolic processes such as denitrification, and thus significantly enhancing denitrification effects. In wastewater treatment and environmental remediation, AHLs (Alternative Hydrological Communities) hold promise for optimizing the control of microbial ecology in complex environments by regulating the structure and function of microbial communities, thereby improving pollutant removal efficiency and system stability. In summary, the novel LuxI protein of this invention not only provides a molecular tool for further elucidating the biological functions of quorum sensing signals, but also offers new solutions and technical support for applications in synthetic biology, agriculture, medicine, and environmental engineering.

[0042] The following description is based on specific implementation methods.

[0043] Example 1: LuxI nucleotide and amino acid sequence prediction Using the QSP database as a reference, candidate sequences were initially screened from metagenomic assembled genomes (MAGs) and non-redundant gene sets in deep-sea cold seep sediments using the HMMER tool (v3.2.1, parameter: cut_ga). Subsequently, MAFFT (v7.505, default parameters) multiple sequence alignment was performed on the candidate LuxI sequences, and TrimAL (v1.4.1, default parameters) sequence trimming was used to optimize alignment quality. A maximum likelihood phylogenetic tree was constructed using FastTree (v2.1.11, default parameters), and the evolutionary relationships of the candidate sequences were visualized and analyzed using iTOL (v6). Furthermore, domain annotation was performed using the Pfam database, and the three-dimensional structure of the LuxI protein was predicted using the AlphaFold2 deep learning tool. Structural similarity clustering was performed using the Foldseek platform, and validity was determined using two thresholds: TM-score > 0.5 and RMSD < 1.

[0044] Following the above steps, among the 3,813 representative MAGs at the cold seep species level, 44 MAGs contained [the following]. luxI Genes; 226 genes were identified from 147 million non-redundant genes in cold seep environments. luxI The gene abundance in different cold seep sediment samples averaged 1.48 GPM (number of genes per million).

[0045] Example 2 Heterologous expression of LuxI protein 1. Sequence processing Prodigal (v2.6.3, parameter: -meta) was used to predict protein-coding sequences (CDSs). Five LuxI proteins containing conserved active sites (R25, F29, W35, D46, D49, R70, E101, R104) were selected for heterologous expression experiments. Their amino acid sequences are as follows: >HM_SQ_cobin_128_LuxI SEQ ID NO: 1:MLIAEKGYRRAGKKITSQIFIKEDSLIVKDMITEHEKIQAFHLRHRIFCQELGWLRQTVNTIETDDYDSKAIFFGVFDRQNNLLAFLRVIMNEAPFMLENDFVSLVMPGYNIRKEDDTIEVSRFCVAPE ARNDIVSENFRACGISMLLYKGVYHWSIKHKKRYLYLVVEYKIYRLLRVRGFPCKLLGDPQKMSDGVIAVAAMIDWREFEALNLSKRPEMLKWFNSLNSKPVTVFNQDQSISPEWRLRRPGAGLRRQAFPE.

[0046] >P_20_sbin.39_LuxI SEQ ID NO: 2: MLTYLYADQLDAYPQLKHSLFKDRASQFRDRLGWDVTVDEQGFEKDKYDAQNPLYVIWRTAQGRHGGSMRVLPTTSPCMMVNDFFSDITGCKISSPLIWESTRFCL APDIGAEAGRISAVLMLAGCEIGLNFGLKHAVGVFDPRMIRIYRILGWSPEIMGVQGKGRDKIYVGLWEFSKTIRLGLAQKAGVSTELSRHWFDRAFCTTTAQQACA.

[0047] >X_3_6_sbin.12_LuxI SEQ ID NO: 3: MIKYIYADALDAFPTLQSGMFKDRAWQFKERLNWDVSVDEHGFESDEYDQINPLYVIYELPDGSHGGSMRILPTTGDTMVNDHFSDLTDGVHISSPLIWECTRFCMSPNAGKLAGKVAASLMLAGCELGIQFGLDSCVGVFDARMISIYKRIGWVPELLGTIGAGKTAISAGIWPIDAESKAQICAKTGIPAIEAENWFEASFPRTDLSDILVA。

[0048] >X_6_9_sbin.73_LuxI SEQ ID NO: 4: MRATTLSFDNMHTHGELFTTLLRTRKQSFVLEHNWDMPVADGMEYDQYDSPASRWIAVHENGNVLGGVRLTPTTAKCGIYSYMIRDAQSGLLDYMPKDLLHFDAPTAPHIWEASRMFISKSVPKNEHAAVKSKLIKELIKTTRVLGATQIIGIMPTIWPIDVESLGIDVAPAGPKVMIDGIEVQVALMIVTSALN。

[0049] >XST_SQ83_0.10_sbin_20_LuxI SEQ ID NO: 5: MTVVCLNWETAHLHGETWISHHRLRHRLFVERQGWDVPSYRGMEHDEFDTPAAQYLVWVDDAGDTRGVARLLPTMQPYMLKKLWPDMISGELPESDSVWEASRFGCDRTLDAPTRRRVVAEILCAMQEFGIRNGIDRYLAVMPLRLLNCVVVDAGCKVTVLGPERAVGNLPAAASYLTVSPEVLAEIRRRASAS。

[0050] The nucleotide sequences of the original coding genes of the above 5 proteins are as follows: >HM_SQ_cobin_128_LuxI SEQ ID NO: 6:ATGCTGATTGCTGAAAAAGGATATCGCAGAGCGGGCAAGAAAATAACGTCTCAAATTTTCATAAAAGAGGACAGCCTTATTGTAAAAGACATGATAACTGAACATGAGAAAATCCAGGCGTTTCACCTGAGACATAGAATATTCTGTCAGGAGCTGGGCTGGCTGCGGCAGACAGTGAATACAATTGAGACGGATGATTATGATTCCAAAGCAATCTTCTTTGGTGTATTTGACCGGCAAAACAATCTATTGGCATTTCTGAGAGTTATAATGAATGAGGCCCCTTTTATGCTGGAAAATGACTTCGTCTCATTGGTTATGCCGGGGTATAACATCAGGAAAGAAGACGATACAATTGAGGTTTCAAGATTTTGTGTCGCGCCTGAGGCAAGGAATGACATTGTCTCTGAAAATTTCAGGGCCTGCGGCATTTCCATGCTTCTTTACAAGGGAGTCTATCACTGGTCGATTAAACATAAAAAAAGATACCTTTATCTGGTAGTAGAATATAAAATATACAGATTGCTTCGTGTAAGAGGCTTTCCCTGTAAGTTATTAGGGGATCCTCAAAAAATGTCTGACGGGGTCATAGCTGTAGCAGCGATGATAGACTGGCGAGAGTTCGAAGCGCTGAATTTGTCAAAGCGGCCCGAGATGCTCAAGTGGTTTAACTCGCTAAACAGTAAACCCGTCACAGTGTTCAATCAAGATCAATCAATCTCTCCTGAATGGCGATTGCGACGGCCTGGGGCCGGCTTACGGCGTCAAGCTTTTCCAGAATAG。

[0051] >P_20_sbin.39_LuxI SEQ ID NO: 7:ATGCTGACTTATCTTTATGCTGACCAATTGGATGCCTACCCACAGCTCAAACATAGTCTATTCAAAGATCGGGCCAGCCAATTTCGTGACCGTTTAGGTTGGGATGTGACAGTCGATGAACAGGGTTTTGAAAAAGATAAATATGATGCACAAAACCCGCTGTATGTGATTTGGCGCACCGCACAGGGTCGCCACGGGGGGTCAATGCGGGTACTTCCGACCACGTCCCCTTGTATGGTTAACGATTTTTTTTCAGATATCACTGGCTGTAAAATCAGCAGCCCCTTGATATGGGAGAGTACACGATTTTGTTTAGCACCTGATATTGGTGCAGAAGCAGGCCGTATTTCAGCAGTGTTAATGCTAGCTGGTTGCGAAATAGGCCTGAATTTTGGCTTAAAACATGCTGTTGGCGTTTTCGATCCGCGAATGATTCGAATTTACCGAATCCTTGGCTGGTCACCTGAAATTATGGGGGTGCAGGGCAAAGGGCGTGATAAAATTTACGTTGGCCTGTGGGAGTTTTCAAAAACCATACGCCTTGGACTGGCCCAGAAAGCAGGTGTTTCAACAGAATTGTCGCGCCACTGGTTTGATCGGGCATTTTGCACAACAACCGCACAACAGGCTTGTGCTTAA。

[0052] >X_3_6_sbin.12_LuxI SEQ ID NO: 8:ATGATAAAATACATCTACGCAGATGCGCTTGACGCATTCCCAACCCTTCAATCCGGTATGTTCAAAGACCGTGCCTGGCAATTCAAAGAGCGCCTGAACTGGGACGTTTCGGTTGATGAACACGGTTTTGAGTCTGATGAATATGACCAGATCAACCCGCTCTACGTGATTTATGAACTGCCCGATGGCAGCCATGGCGGGTCAATGCGCATTTTGCCGACAACCGGCGACACAATGGTTAATGACCATTTCAGCGACCTGACCGACGGCGTTCACATATCCAGCCCGTTGATCTGGGAATGTACCCGTTTTTGCATGTCGCCCAACGCCGGCAAGCTGGCAGGCAAGGTTGCGGCATCACTGATGTTGGCAGGTTGTGAACTTGGCATCCAGTTTGGTCTGGACAGCTGTGTTGGCGTTTTTGATGCGCGGATGATCAGCATCTACAAACGTATTGGTTGGGTGCCCGAGTTGCTGGGGACGATTGGCGCGGGAAAAACGGCGATCAGTGCCGGAATCTGGCCAATTGACGCGGAATCAAAGGCGCAGATTTGCGCAAAAACCGGCATTCCGGCGATTGAAGCTGAAAACTGGTTCGAGGCATCGTTCCCGCGTACTGATCTTTCCGATATTCTGGTTGCCTGA。

[0053] >X_6_9_sbin.73_LuxI SEQ ID NO: 9:ATGCGAGCGACTACACTATCTTTTGATAATATGCACACACATGGTGAATTATTCACCACTCTTTTACGCACCCGTAAACAGTCTTTCGTTCTTGAACATAACTGGGATATGCCGGTTGCTGATGGAATGGAATATGACCAATACGACTCCCCTGCAAGCCGCTGGATTGCGGTTCATGAAAACGGAAACGTTCTGGGCGGAGTCCGGTTAACTCCGACTACAGCAAAATGCGGGATTTATTCCTATATGATCCGAGACGCACAAAGTGGCCTACTGGATTACATGCCTAAAGATCTACTGCATTTTGACGCACCGACGGCACCACATATTTGGGAAGCCTCCCGGATGTTTATTTCAAAGTCTGTTCCAAAGAATGAACACGCGGCTGTTAAATCAAAATTGATAAAAGAGCTCATCAAAACAACGCGGGTATTGGGTGCTACACAAATAATCGGCATTATGCCTACTATATGGCCAATTGACGTTGAGAGCCTTGGAATTGATGTGGCACCTGCGGGCCCGAAAGTAATGATAGACGGAATTGAAGTTCAGGTAGCATTGATGATAGTTACATCAGCTCTCAACTAG。

[0054] >XST_SQ83_0.10_sbin_20_LuxI SEQ ID NO: 10:

[0055] The online tool TMHMM was used to predict the transmembrane translocation of candidate LuxI proteins. All five proteins were found to be non-transmembrane translocation proteins. SignalP was used to predict the signal peptide, and none of the five protein sequences contained a signal peptide.

[0056] 2. Gene synthesis The processed gene sequences were handed over to Sangon Biotech (Shanghai) Co., Ltd. for gene synthesis and recombinant expression vector construction. This mainly included: 1) Codon optimization: adjusting codon usage preferences while maintaining the amino acid sequence to better suit efficient expression in *E. coli*; 2) Primer synthesis: using Oligo chemical synthesis, small primer fragments of 40-200 nt with overlapping regions were synthesized; 3) Oligonucleotide splicing: using overlap PCR technology, single-stranded primers were assembled into double-stranded gene fragments; 4) Recombinant expression vector construction: the synthesized DNA fragments were inserted into the pET-28(a) vector using enzyme digestion and assembly technology. After ligation transformation, cloning screening, sequencing verification, and mutation repair, a recombinant vector with a completely correct sequence was obtained. The final quality control of the product was performed through sequencing verification, enzyme digestion, and electrophoresis to obtain recombinant plasmid vectors with correct sequences for subsequent experiments. The structures of the successfully constructed recombinant plasmid vectors encoding genes with optimized codons HM_SQ_cobin_128_LuxI, >P_20_sbin.39_LuxI, >X_3_6_sbin.12_LuxI, >X_3_6_sbin.12_LuxI, >X_6_9_sbin.73_LuxI, and >XST_SQ83_0.10_sbin_20_LuxI are shown below. Figure 1 , Figure 2 , Figure 3 , Figure 4 and Figure 5 As shown.

[0057] The nucleotide sequences of the encoding genes for the above five proteins, after codon optimization by Sangon Biotech (Shanghai) Co., Ltd., are as follows: >HM_SQ_cobin_128_LuxI SEQ ID NO: 11:CATATGCTGATTGCTGAAAAAGGATATCGCAGAGCGGGCAAGAAAATAACGTCTCAAATTTTCATAAAAGAGGACAGCCTTATTGTAAAAGACATGATAACTGAACATGAGAAAATCCAGGCGTTTCACCTGAGACATAGAATATTCTGTCAGGAGCTGGGCTGGCTGCGGCAGACAGTGAATACAATTGAGACGGATGATTATGATTCCAAAGCAATCTTCTTTGGTGTATTTGACCGGCAAAACAATCTATTGGCATTTCTGAGAGTTATAATGAATGAGGCCCCTTTTATGCTGGAAAATGACTTCGTCTCATTGGTTATGCCGGGGTATAACATCAGGAAAGAAGACGATACAATTGAGGTTTCAAGATTTTGTGTCGCGCCTGAGGCAAGGAATGACATTGTCTCTGAAAATTTCAGGGCCTGCGGCATTTCCATGCTTCTTTACAAGGGAGTCTATCACTGGTCGATTAAACATAAAAAAAGATACCTTTATCTGGTAGTAGAATATAAAATATACAGATTGCTTCGTGTAAGAGGCTTTCCCTGTAAGTTATTAGGGGATCCTCAAAAAATGTCTGACGGGGTCATAGCTGTAGCAGCGATGATAGACTGGCGAGAGTTCGAAGCGCTGAATTTGTCAAAGCGGCCCGAGATGCTCAAGTGGTTTAACTCGCTAAACAGTAAACCCGTCACAGTGTTCAATCAAGATCAATCAATCTCTCCTGAATGGCGATTGCGACGGCCTGGGGCCGGCTTACGGCGTCAAGCTTTTCCAGAATAGGAATTC。

[0058] >P_20_sbin.39_LuxI SEQ ID NO: 12:GGATCCATGCTGACTTATCTTTATGCTGACCAATTGGATGCCTACCCACAGCTCAAACATAGTCTATTCAAAGATCGGGCCAGCCAATTTCGTGACCGTTTAGGTTGGGATGTGACAGTCGATGAACAGGGTTTTGAAAAAGATAAATATGATGCACAAAACCCGCTGTATGTGATTTGGCGCACCGCACAGGGTCGCCACGGGGGGTCAATGCGGGTACTTCCGACCACGTCCCCTTGTATGGTTAACGATTTTTTTTCAGATATCACTGGCTGTAAAATCAGCAGCCCCTTGATATGGGAGAGTACACGATTTTGTTTAGCACCTGATATTGGTGCAGAAGCAGGCCGTATTTCAGCAGTGTTAATGCTAGCTGGTTGCGAAATAGGCCTGAATTTTGGCTTAAAACATGCTGTTGGCGTTTTCGATCCGCGAATGATTCGAATTTACCGAATCCTTGGCTGGTCACCTGAAATTATGGGGGTGCAGGGCAAAGGGCGTGATAAAATTTACGTTGGCCTGTGGGAGTTTTCAAAAACCATACGCCTTGGACTGGCCCAGAAAGCAGGTGTTTCAACAGAATTGTCGCGCCACTGGTTTGATCGGGCATTTTGCACAACAACCGCACAACAGGCTTGTGCTTAAGAATTC。

[0059] >X_3_6_sbin.12_LuxI SEQ ID NO: 13:GGATCCATGATAAAATACATCTACGCAGATGCGCTTGACGCATTCCCAACCCTTCAATCCGGTATGTTCAAAGACCGTGCCTGGCAATTCAAAGAGCGCCTGAACTGGGACGTTTCGGTTGATGAACACGGTTTTGAGTCTGATGAATATGACCAGATCAACCCGCTCTACGTGATTTATGAACTGCCCGATGGCAGCCATGGCGGGTCAATGCGCATTTTGCCGACAACCGGCGACACAATGGTTAATGACCATTTCAGCGACCTGACCGACGGCGTTCACATATCCAGCCCGTTGATCTGGGAATGTACCCGTTTTTGCATGTCGCCCAACGCCGGCAAGCTGGCAGGCAAGGTTGCGGCATCACTGATGTTGGCAGGTTGTGAACTTGGCATCCAGTTTGGTCTGGACAGCTGTGTTGGCGTTTTTGATGCGCGGATGATCAGCATCTACAAACGTATTGGTTGGGTGCCCGAGTTGCTGGGGACGATTGGCGCGGGAAAAACGGCGATCAGTGCCGGAATCTGGCCAATTGACGCGGAATCAAAGGCGCAGATTTGCGCAAAAACCGGCATTCCGGCGATTGAAGCTGAAAACTGGTTCGAGGCATCGTTCCCGCGTACTGATCTTTCCGATATTCTGGTTGCCTGAGAATTC。

[0060] >X_6_9_sbin.73_LuxI SEQ ID NO: 14:GGATCCATGCGAGCGACTACACTATCTTTTGATAATATGCACACACATGGTGAATTATTCACCACTCTTTTACGCACCCGTAAACAGTCTTTCGTTCTTGAACATAACTGGGATATGCCGGTTGCTGATGGAATGGAATATGACCAATACGACTCCCCTGCAAGCCGCTGGATTGCGGTTCATGAAAACGGAAACGTTCTGGGCGGAGTCCGGTTAACTCCGACTACAGCAAAATGCGGGATTTATTCCTATATGATCCGAGACGCACAAAGTGGCCTACTGGATTACATGCCTAAAGATCTACTGCATTTTGACGCACCGACGGCACCACATATTTGGGAAGCCTCCCGGATGTTTATTTCAAAGTCTGTTCCAAAGAATGAACACGCGGCTGTTAAATCAAAATTGATAAAAGAGCTCATCAAAACAACGCGGGTATTGGGTGCTACACAAATAATCGGCATTATGCCTACTATATGGCCAATTGACGTTGAGAGCCTTGGAATTGATGTGGCACCTGCGGGCCCGAAAGTAATGATAGACGGAATTGAAGTTCAGGTAGCATTGATGATAGTTACATCAGCTCTCAACTAGGAATTC。

[0061] >XST_SQ83_0.10_sbin_20_LuxI SEQ ID NO: 15:

[0062] 3. Bacterial culture The recombinant expression plasmid with the correct sequence and the empty pET-28(a) plasmid were transformed into *E. coli* BL21(DE3) competent cells, respectively. After transformation, the cells were plated on LB agar plates containing kanamycin (50 µg / mL) and incubated overnight (approximately 12 h) at 37°C to obtain single colonies (target positive colonies). A single colony was picked from the plate and inoculated into 5 mL of LB liquid medium containing kanamycin (50 µg / mL) and cultured with shaking in a shaking incubator to obtain a seed culture. A 1% (v / v) bacterial culture was then inoculated into LB liquid medium containing kanamycin (50 µg / mL) and cultured with shaking (culture conditions: 20°C, 150 rpm).

[0063] 4. Induced expression When the optical density (OD600) of the culture reaches about 0.5, add 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and continue to incubate overnight (culture conditions: 16°C, 150 rpm).

[0064] 5. Collect the bacterial cells and store them in a -80°C freezer.

[0065] 6. Ultrasonic fragmentation The collected bacterial cells were resuspended in 50 mM phosphate buffer and disrupted by ultrasonic lysis (5 s sonication, 20 s interval, 36% power, 3 min total time) to obtain the lysate.

[0066] 7. SDS-PAGE Analysis Lysis buffer was analyzed by 10% SDS-PAGE. Compared with the blank control (E. coli lysate transformed with pET-28(a) vector without the target gene), the experimental group showed obvious bands at the corresponding molecular weights, indicating that the five proteins were successfully expressed heterologously. Figure 6 ).

[0067] Example 3: Validation of LuxI protein activity 1. Indicator bacteria culture Will Agrobacterium tumefaciens A136 strain was inoculated into LB liquid medium containing kanamycin (50 µg / mL) and cultured overnight (culture conditions: 30°C, 200 rpm).

[0068] 2. Coating Take 100 μL of fresh culture medium and spread it evenly on an LB agar plate containing kanamycin (50 µg / mL) using the pour plate method.

[0069] 3. Cover with hydrogel After incubation at 30°C for 24 h, an X-gal hydrogel layer with a final concentration of 40 µg / L was coated onto the plate surface.

[0070] 4. Drill holes and add pyrolysis solution After the hydrogel solidified, six wells were punched, and 50 μL of the positive expression bacterial lysis buffer from Example 2 was added to five of the wells, with one well serving as a blank control. The wells were then sealed with hydrogel. The cells were incubated at room temperature for another 24 h.

[0071] 5. Color reaction Compared with the control group, the lysates of four LuxI-expressing strains (>HM_SQ_cobin_128_LuxI, >P_20_sbin.39_LuxI, >X_3_6_sbin.12_LuxI, and >X_6_9_sbin.73_LuxI) significantly induced a blue reaction in the biosensor bacteria during plate punch detection. Figure 7 Furthermore, the four LuxI proteins exhibited stable and reproducible signals under X-gal coverage, demonstrating that they can all synthesize and secrete acylhomoserine lactones (AHLs) molecules.

[0072] The structure predictions and structure comparisons for >HM_SQ_cobin_128_LuxI, >P_20_sbin.39_LuxI, >X_3_6_sbin.12_LuxI, and >X_6_9_sbin.73_LuxI are as follows: Figure 8 As shown.

[0073] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments 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.

[0074] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. An isolated protein, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:

4.

2. An isolated polynucleotide, characterized in that, The polynucleotide encodes the protein as described in claim 1.

3. The polynucleotide as described in claim 2, characterized in that, The nucleotide sequence of the polynucleotide encoding the protein with the amino acid sequence shown in SEQ ID NO: 1 is shown in SEQ ID NO: 6 or SEQ ID NO: 11; The nucleotide sequence of the polynucleotide encoding the protein with the amino acid sequence shown in SEQ ID NO: 2 is shown in SEQ ID NO: 7 or SEQ ID NO: 12; The nucleotide sequence of the polynucleotide encoding the protein with the amino acid sequence shown in SEQ ID NO: 3 is shown in SEQ ID NO: 8 or SEQ ID NO: 13; The nucleotide sequences of the polynucleotides encoding the protein with the amino acid sequence shown in SEQ ID NO: 4 are shown in SEQ ID NO: 9 or SEQ ID NO:

14.

4. A recombinant expression vector, characterized in that, The recombinant expression vector is inserted with the polynucleotide as described in any one of claims 2 to 3.

5. The recombinant expression vector as described in claim 4, characterized in that, The backbone vector of the recombinant expression vector is pET-28(a).

6. A recombinant engineered bacterium, characterized in that, The recombinant engineered bacteria comprises the protein as described in claim 1, the polynucleotide as described in any one of claims 2 to 3, or the recombinant expression vector as described in any one of claims 4 to 5.

7. The recombinant engineered bacteria as described in claim 6, characterized in that, The host bacterium of the recombinant engineered bacteria is Escherichia coli, preferably Escherichia coli BL21(DE3).

8. The use of the protein of claim 1, the polynucleotide of any one of claims 2-3, the recombinant expression vector of any one of claims 4-5, and / or the recombinant engineered bacteria of any one of claims 6-7 in the preparation of acylhomoserine lactone.

9. A method for preparing acylhomoserine lactone, characterized in that, Includes the following steps: The protein as described in claim 1 is used as a synthase to catalyze the synthesis of acylhomoserine lactone.

10. The method as described in claim 9, characterized in that, The process includes the following steps: (1) culturing the recombinant engineered bacteria as described in any one of claims 6 to 7; (2) adding an inducer to the culture system and continuing the culture; (3) collecting, breaking, and separating the bacterial cells to obtain the final product. Preferably, the inducing agent is isopropyl-β-D-thiogalactopyranoside; and / or, The method of crushing is ultrasonic crushing.

Images