Use of promoter libraries in genetic transformation and gene expression in rumenococci

Abstract

The present application relates to the technical field of biotechnology, and particularly relates to the application of a promoter library in genetic transformation and gene expression of enterococcus bacteria, wherein the promoter library comprises promoters with nucleotide sequences as shown in SEQ ID No. 1-SEQ ID No. 15. The promoter library comprising promoters with nucleotide sequences as shown in SEQ ID No. 1-SEQ ID No. 15 has good adaptability and stability, and can be applied to genetic transformation and gene expression research of various enterococcus bacteria.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to the application of promoter libraries in the genetic transformation and gene expression of enterorumenococci. Background Technology

[0002] *Ruminococcus gnavus* is a core and ubiquitous member of the human gut microbiota. Studies have shown that changes in the abundance of *Ruminococcus gnavus* are closely associated with various diseases, including inflammatory bowel disease, obesity, metabolic syndrome, and neurological disorders. *Ruminococcus gnavus* belongs to the genus *Ruminococcus*, which consists of typically strictly anaerobic, Gram-positive cocci that rely on fermentable carbohydrates for growth and is one of the most abundant microbiota in the mammalian gut environment. The genus *Ruminococcus* includes several intestinal colonizing species, primarily including *Ruminococcus torques*, *Ruminococcus lactaris*, and *Ruminococcus faecis*. Intestinal colonization by *Ruminococcus* bacteria is closely related to various human diseases; changes in their abundance or abnormal proliferation of specific strains are associated with digestive tract diseases, metabolic diseases, immune diseases, neurological diseases, and liver diseases.

[0003] Genetic transformation systems are fundamental to microbial genetic engineering research and genetic manipulation, with the core being the efficient introduction, stable maintenance, and controlled expression of exogenous DNA in host cells. Current technologies for genetic manipulation systems targeting active rumenococci and related rumenococcal bacteria still suffer from low transformation efficiency and insufficient stability. Promoters, as key elements regulating gene expression, can precisely regulate the intensity and timing of downstream gene expression by modulating transcription initiation efficiency. With the development of transcriptomics technology, systematic screening of endogenous constitutive promoters in microbial genomes based on whole-genome transcriptional data can yield constitutive promoter elements with different expression levels. Compared to exogenous promoters, naturally derived promoter sequences typically exhibit better host adaptability and expression stability, making them ideal gene expression regulatory elements. However, current technologies lack promoter libraries capable of covering different expression intensity gradients, making it difficult to meet the needs of gene expression regulation and metabolic engineering in these strains. Summary of the Invention

[0004] The main objective of this invention is to provide an application of a promoter library in the genetic transformation and gene expression of enterorumen cocci, in order to solve at least one of the above-mentioned technical problems.

[0005] The inventors of this invention optimized the genetic transformation system to obtain a system particularly suitable for *Ruminococcus* bacteria and capable of maintaining a high level of genetic transformation efficiency for these bacteria. Using this system, the inventors further constructed a promoter library containing promoters with nucleotide sequences as shown in SEQ ID No. 1-SEQ ID No. 15. Verification experiments showed that the promoters in this library not only cover different expression intensity gradients but are also applicable to a variety of different *Ruminococcus* bacteria, demonstrating a certain degree of versatility.

[0006] According to a first aspect of the present invention, a promoter library comprising promoters with nucleotide sequences as shown in SEQ ID No. 1-SEQ ID No. 15 is provided for use in genetic transformation and gene expression of enterorumenococci.

[0007] The promoters in the promoter library provided by this invention have gradient gene expression intensity, providing host bacteria with endogenous expression regulatory elements with good adaptability and stability. Furthermore, the promoter library provided by this invention is applicable to a variety of enterorumen cocci and has a certain degree of versatility.

[0008] In some embodiments, the enteric rumenococci may be active rumenococci, streptococci, lactococci, or fecal rumenococci.

[0009] In some embodiments, the promoter library provided by the present invention can be used for recombinant strain construction, exogenous gene expression, endogenous gene overexpression, metabolic pathway optimization, reporter system construction, or synthetic biology tool development.

[0010] According to a second aspect of the present invention, a recombinant vector is provided, which contains a promoter with nucleotide sequences as shown in any of SEQ ID No. 1-SEQ ID No. 15. The recombinant vector provided by the present invention is constructed by inserting the promoter and the target gene into the pMTL83151 plasmid as a backbone.

[0011] The recombinant vector provided by the present invention contains a promoter with nucleotide sequences as shown in any of SEQ ID No. 1-SEQ ID No. 15. Therefore, recombinant vectors that characterize different promoter strengths can be flexibly selected as needed.

[0012] In some implementations, homologous recombination is used to insert the promoter and target gene into the plasmid to construct a recombinant vector.

[0013] According to a third aspect of the present invention, a recombinant strain is provided, which is obtained by introducing the recombinant vector provided by the present invention into a host bacterium, wherein the host bacterium is an enterorumenococcus including active rumenococcus, torsion rumenococcus, lactobacillus, and fecal rumenococcus, thereby enabling the universal use of the recombinant vector provided by the present invention in a variety of enterorumenococcus bacteria.

[0014] According to a fourth aspect of the invention, the application of recombinant vectors or recombinant strains in the genetic transformation and gene expression of enterorumenococci is provided.

[0015] The promoter library, recombinant vector, or recombinant strain provided by this invention can be universally used in a variety of enterorumenococcal bacteria for gene expression regulation. Attached Figure Description

[0016] Figure 1 The graph shows the results of screening the conjugation efficiency of shuttle plasmids with Escherichia coli donor bacteria in this invention. Figure 2 The figure shows the conjugation efficiency results of optimizing the conjugation and transfer co-culture time according to the present invention; Figure 3 The graph shows the conjugation efficiency results of optimizing the donor bacterial addition amount in this invention. Figure 4 The diagram shows the results of optimizing the binding efficiency of the recipient bacteria's optical density (OD600) according to this invention. Figure 5 The diagram shows the conjugation efficiency results of optimizing the optical density (OD600) of the donor bacteria in this invention. Figure 6 This is a map of the promoter gene expression vector of the present invention; Figure 7 This is a promoter characterization diagram of the promoter library of the present invention; Figure 8 This is a graph showing the conjugation efficiency results of the genetic transformation system of the present invention in *Ruminococcus truncatula*, *Ruminococcus lactis* and *Ruminococcus fetus*. Figure 9 Characterization diagrams of promoters P00815, P08965, and P10570 applied to *Ruminococcus truncatula*, *Ruminococcus lactis*, and *Ruminococcus fecalis*. Detailed Implementation

[0017] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following examples are all commercially available.

[0018] Experimental Example 1 Constructing a genetic transformation system includes the following steps: S1. Incubate Enterococcus spp. in BHI-YH liquid medium at 37°C for overnight anaerobic culture, and adjust the bacterial concentration to the required OD600 value to form an overnight culture of Enterococcus spp. The shuttle plasmid was transformed into donor bacteria using the heat shock method to obtain donor bacteria containing the shuttle plasmid. Donor bacteria containing shuttle plasmids were cultured overnight at 37°C in LB liquid medium. The bacterial concentration was adjusted to the required OD600 value. Subsequently, a specific volume of E. coli culture was centrifuged and washed twice with BHI-YH liquid medium to obtain a precipitate. S2. Under anaerobic conditions, the obtained precipitate was resuspended in a specific volume of overnight culture of Ruminococcus spp., mixed well, and then spotted onto a co-culture medium. The conjugation time was 37°C to obtain conjugated bacterial sludge. The co-culture medium was BHI-YH solid medium. S3. Resuspend the conjugating bacteria sludge in 1 mL of BHI-YH liquid medium, then spread it on BHI-YH solid medium containing 250 μg / mL D-cyclic serine and 15 μg / mL thiamphenicol, and culture anaerobically at 37°C to obtain conjugators carrying shuttle plasmids.

[0019] 1. Screening of shuttle plasmids and Escherichia coli donor bacteria Recipient bacteria: Active rumenococci in the genus Ruminococcus; Shuttle plasmids: selected from the pMTL80000 series shuttle plasmids, namely pMTL82151, pMTL83151, pMTL84151, and pMTL85151. All of the above plasmids contain the catP gene. Donor bacteria: Escherichia coli CA434 and Escherichia coli S17, respectively, with Escherichia coli CA434 carrying the KanR gene.

[0020] 1.1 Construction of recombinant donor strains (1) The above four shuttle plasmids were transformed into competent cells of two donor bacteria to obtain recombinant cells; (2) The obtained recombinant cells were seeded on LB solid medium plates (containing 30 μg / mL chloramphenicol) and cultured overnight at 37°C; (3) Pick individual clones and inoculate them into LB liquid medium (containing 30 μg / mL chloramphenicol); (4) Use the verification primers to perform PCR verification. If the result meets the expectations, send it for sequencing. If the sequencing is correct, the recombinant strain of the donor bacteria is obtained.

[0021] The validation primers used are shown in Table 1.

[0022] Table 1

[0023] 1.2 Construction of recombinant active rumenococcal strains According to the steps of constructing a genetic transformation system, the corresponding shuttle plasmid was introduced into active rumenococci. Single clones were picked on BHI-YH solid medium containing the corresponding antibiotics and inoculated into BHI-YH liquid medium (containing 250 μg / mL D-cyclic serine and 15 μg / mL thiamphenicol).

[0024] PCR verification was performed using the verification primers shown in Table 1, as well as the universal primers 16S-27F and 16S-1492R for the 16S rRNA gene. After the results met expectations, the 16S rRNA PCR products were sequenced, and the sequencing results were compared with BLAST sequences.

[0025] Resistance screening and plasmid stability verification revealed that among the tested pMTL80000 series shuttle plasmids, only pMTL83151 plasmid could stably replicate in active rumenococci. Furthermore, compared to donor strain S17, using *Escherichia coli* CA434 as the donor strain significantly improved conjugation transfer efficiency. The conjugation efficiency results are as follows: Figure 1 As shown above, the results indicate that the combination of pMTL83151 and Escherichia coli CA434 is more suitable for the conjugation transformation system of active rumenococci.

[0026] 2. Optimization of key influencing factors in bonding transfer The shuttle plasmid used was confirmed to be pMTL83151, the donor bacteria was Escherichia coli CA434, and the recipient bacteria was active Ruminococcus, with a recipient bacteria volume of 200 μL.

[0027] Influencing parameters: Co-cultivation (conjugation) time: 8h, 12h, 16h; Donor bacteria addition amounts: 1 mL, 3 mL, 5 mL; Recipient bacterial optical density (OD600): 0.5, 1.0, 2.0; Donor bacterial optical density (OD600): 0.5, 1.0, 2.0; Based on the selection of the aforementioned influencing parameters, a single-factor variable method was adopted. This involved keeping other conditions constant and changing only one experimental parameter, following the construction method of the genetic transformation system described above. Transformation efficiency was assessed by counting the number of conjugates through antibiotic selection and PCR verification. The conjugation efficiency results for different influencing parameters are shown below. Figures 2 to 5As shown, the experimental results indicate that different parameters have a significant impact on conjugation transfer efficiency. By systematically optimizing the above conditions, the genetic transformation efficiency of active rumenococci can be significantly improved.

[0028] The optimal parameters were 8 h for conjugation transfer co-culture, 2.0 OD600 value of recipient bacteria, 1.0 OD600 value of donor bacteria, and 1 mL of donor bacteria added.

[0029] Example 1 A method for constructing a genetic transformation system suitable for enteric rumenococci includes the following steps: S1. Active rumenococci are cultured anaerobically overnight at 37°C in BHI-YH liquid medium, and the bacterial concentration is adjusted to an OD600 value of 2.0 to form a culture medium of active rumenococci. Remove E. coli CA434 competent cells from a -80°C environment, thaw them on ice and mix them gently. 10 ng of pMTL83151 plasmid was added to 50 μL of Escherichia coli CA434 competent cells, mixed thoroughly, and then incubated on ice for 30 min. The mixture was heat-shocked at 42°C for 90 seconds and then immediately transferred to an ice bath to cool for 2 minutes. Add 1 mL of antibiotic-free LB liquid medium to the mixture and shake to recover for 60 min at 37°C and 250 rpm. After resuscitation, centrifuge at 4000g for 7 minutes to remove all supernatant and obtain bacterial cells; After resuspending the bacterial cells, they were evenly spread on LB solid plates containing the resistant bacteria and incubated overnight at a constant temperature. Escherichia coli CA434 containing the pMTL83151 plasmid was selected. Escherichia coli CA434 containing pMTL83151 plasmid was cultured overnight at 37°C in LB liquid medium. The bacterial concentration was adjusted to an OD600 value of 1.0 to obtain Escherichia coli culture. Subsequently, 1 mL of Escherichia coli culture was centrifuged and washed twice with BHI-YH liquid medium to obtain a precipitate. S2. Under anaerobic conditions, the obtained precipitate was resuspended in 200 μL of active rumenococcal culture medium, mixed well, and then spotted onto BHI-YH solid medium for co-culture. The mixture was then co-cultured at 37°C for 8 h to obtain conjugated bacterial sludge. S3. Using 1 mL of BHI-YH liquid medium, scrape off the zygote mud and spread it on BHI-YH solid medium containing the corresponding antibiotic. Incubate anaerobically at 37°C to obtain zygotes carrying shuttle plasmids.

[0030] Transformation efficiency was assessed by counting the number of conjugates through resistance selection and PCR verification. Under this highly efficient genetic transformation system, the conjugation efficiency could reach 2.7 × 10⁻⁶. -5 .

[0031] Example 2 The method for building a sub-library includes the following steps: 1. Screening promoters based on transcriptome data Active rumenococci were inoculated into BHI-YH medium and cultured anaerobically at 37°C. Samples were collected during the lag phase, logarithmic growth phase, and stationary phase of bacterial growth for transcriptome sequencing analysis. Based on the measured transcriptome data, FPKM value was used as an evaluation index for gene expression levels. Candidate constitutive genes were screened according to gene expression intensity and expression stability analysis. High-expression genes (FPKM > 10000), medium-expression genes (4000 ≤ FPKM ≤ 8000), and low-expression genes (1000 ≤ FPKM ≤ 2000) were screened. Finally, 15 genes with stable expression and no significant differential expression were selected as candidate genes. The upstream sequence of the start codon of the candidate genes was extracted as candidate promoters; the 250 bp upstream sequence of the start codon of the candidate genes was extracted as candidate promoter fragments for subsequent promoter functional characterization and promoter library construction. The 15 candidate promoters and their corresponding FPKM values ​​are shown in Table 2.

[0032] Table 2

[0033] 2. Construction of promoter recombination vector Genomic DNA was extracted from active rumenococci using the EasyPure Bacteria Genomic DNA Kit from Beijing TransGen Biotechnology Co., Ltd. Primers were designed to amplify candidate promoter sequences using this genomic DNA as a template. The gusA gene was also selected as a reporter gene, and primers were designed for its amplification. The sequences of the primers used for amplifying the candidate promoter and the gusA gene are shown in Table 3.

[0034] Table 3

[0035] All PCR amplification reactions were performed using Phanta Max Super-Fidelity DNA Polymerase, a high-fidelity DNA polymerase. The PCR reaction system is shown in Table 4, and the reaction procedure is shown in Table 5.

[0036] Table 4

[0037] Table 5

[0038] The obtained PCR products were purified using the Omega EZNA Gel Extraction Kit to obtain candidate promoter fragments and reporter gene fragments.

[0039] Using plasmid pMTL83151 as the backbone, pMTL83151 was double-digested with NdeⅠ and HindⅢ to purify and obtain the linearized pMTL83151 vector backbone. Using the ClonExpress Ultra One Step Cloning Kit, following the instructions, the candidate promoter fragment, reporter gene fragment, and linearized pMTL83151 vector backbone were added to the homologous recombination system and incubated to obtain the recombination reaction mixture. After the reaction was completed, the recombinant reaction mixture was transformed and introduced into Escherichia coli DH5α competent cells, and single-clone colonies were obtained by culturing. Single colonies were picked and PCR was performed using the validation primers p83-li-F and GusA-li-R, as well as GusA-p83-li-F and GusA-p83-li-R. The primer sequences are shown in Table 6.

[0040] Table 6

[0041] Candidate positive strains that perfectly match the theoretical expected PCR amplification band size are screened and sent for sequencing. Recombinant plasmids are extracted from the correctly sequenced strains using the Omega EZNA Plasmid DNA Mini KitⅠ, yielding gene expression vectors carrying promoters with different expression intensities. Specifically, the promoter gene expression vector map is shown below. Figure 6 As shown.

[0042] 3. Construction of recombinant donor strains The promoter gene expression vector described above was transformed into Escherichia coli CA434, and PCR verification was performed using the verification primers p83-li-F and GusA-li-R. The primer sequences are shown in Table 6.

[0043] 4. Construction of recombinant strains According to the method for constructing the genetic transformation system described in Example 1, the promoter gene expression vector was introduced into the host bacterium *Ruminococcus rumeniculatus*. Conjugates were picked from BHI-YH solid medium containing the corresponding antibiotics and transferred to BHI-YH liquid medium containing a final concentration of 250 μg / mL D-cyclic serine and 15 μg / mL thiamphenicol for secondary screening. PCR verification was performed using validation primers p83-li-F and GusA-li-R, and universal primers 16s-27F and 16s-1492R. The specific primer sequences are shown in Table 6. After the verification results met expectations, the samples were sequenced. The correctly sequenced recombinant strains were grown to an OD600 of 1.0, and 1.5 mL of the recombinant strain sample was harvested by centrifugation and stored at -80°C.

[0044] 5. Expression level of the startup sub-library The recombinant bacterial strain sample was resuspended in 1 mL of lysis buffer Z. 400 μL of the sample was used for OD600 detection. 6 μL of toluene was added to the remaining sample. After pre-incubation at 37 °C for 30 min, 120 μL of 6 mM p-nitrophenyl-β-D-glucuronide was added to initiate the reaction. The parameters of lysis buffer Z are shown in Table 7.

[0045] Table 7

[0046] After incubating at 37°C for 5-30 min, 300 μL of 1M Na2CO3 was added to terminate the reaction, and the reaction time was recorded. Then, the cells were centrifuged at 10000g for 10 min at room temperature to remove cell debris. The supernatant was transferred to a polystyrene spectrophotometer cuvette, and the absorbance at 405 nm was measured.

[0047] The glucuronidase activity was calculated by dividing the absorbance at 405 nm by the absorbance at 600 nm and the incubation time, and then normalized to the GusA activity of the wild-type strain (WT). The results are as follows: Figure 7 As shown.

[0048] Based on the expression intensity of different candidate promoters, they are classified and categorized to form a promoter library covering different expression intensity gradients.

[0049] Example 3 Application of genetic transformation systems suitable for enteric rumenococci. Using the genetic transformation system for enteric rumenococci described in Example 1, the recombinant plasmid from the Escherichia coli CA434 donor recombinant strain containing the pMTL83151 plasmid was transferred into R. torques, R. lactaris, and R. faecis.

[0050] Single clones were picked and inoculated into BHI-YH liquid medium (containing 250 μg / mL D-cyclic serine and 15 μg / mL thiamphenicol).

[0051] PCR verification was performed using the validation primers shown in Table 1 and the universal primers 16S-27F and 16S-1492R for the 16S rRNA gene. After confirming the expected results, the 16S rRNA PCR product was sequenced. BLAST sequence alignment was performed on the sequencing results, showing that the genetic transformation system provided by this invention was successfully applied to the aforementioned *Ruminococcus* bacteria. The results are as follows: Figure 8 As shown, in addition to active rumenococci, the genetic transformation system provided by this invention can also be applied to other intestinal rumenococci such as *Ruminococcus tortifolius*, *Ruminococcus lactis*, and *Ruminococcus fecalis*.

[0052] Example 4 Application of the promoter library provided by this invention Three promoters, P00815, P08965, and P10570, were selected. Using pMTL83151 plasmid as the backbone and the β-glucuronidase gene (GusA) as the reporter gene, the promoters and reporter gene were inserted into the plasmid backbone to form recombinant plasmids p83-P00815-GusA, p83-P08965-GusA, and p83-P10570-GusA, respectively. Using the genetic transformation system applicable to enteric rumenococci as described in Example 1, the resulting recombinant plasmids were transformed into Escherichia coli CA434 to obtain recombinant Escherichia coli CA434 strains containing the corresponding recombinant plasmids. The recombinant plasmids from the recombinant strains were then transferred into R. torques, R. lactaris, and R. faecis.

[0053] PCR verification was performed using validation primers p83-li-F and GusA-li-R, as well as universal primers 16s-27F and 16s-1492R. Primer sequences are shown in Table 6.

[0054] After meeting the expected results, the samples were sent for sequencing. The correctly sequenced recombinant strains were then characterized, and the results were as follows: Figure 9As shown, in addition to active rumenococci, the genetic transformation system provided by this invention can also be applied to other intestinal rumenococci such as *Ruminococcus tortifolius*, *Ruminococcus lactis*, and *Ruminococcus fecalis*.

[0055] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. The application of promoter libraries in the genetic transformation and gene expression of *Ruminococcus* spp., characterized in that, The promoter library includes promoters with nucleotide sequences as shown in SEQ ID No. 1-SEQ ID No.

15.

2. A recombinant vector, characterized in that, Contains a promoter with a nucleotide sequence as shown in any of SEQ ID No. 1-SEQ ID No. 15; The recombinant vector was constructed by inserting the promoter and target gene into the pMTL83151 plasmid as a backbone.

3. A recombinant bacterial strain, characterized in that, The recombinant strain is obtained by introducing the recombinant vector of claim 2 into a host bacterium.

4. The application of the recombinant vector of claim 2 and the recombinant strain of claim 3 in the genetic transformation and gene expression of enterorumenococci.

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