A method and application for improving lincomycin production by modifying the SLCG_1608 gene of Streptomyces lincomyces.

Abstract

This invention relates to the field of genetic engineering technology and provides a method for increasing lincomycin production by modifying the SLCG_1608 gene in *Streptomyces lincomyces*. The method involves overexpressing the Lrp family transcriptional regulatory gene SLCG_1608 in *Streptomyces lincomyces* through genetic engineering, obtaining a high-yielding lincomycin-producing strain, which is then used for fermentation to produce lincomycin. The nucleotide sequence of the SLCG_1608 gene is shown in SEQ ID NO.1. This invention also provides the application of the above method in industrial strains. The advantages of this invention are: increasing the copy number of the SLCG_1608 gene on the chromosome of *Streptomyces lincomyces* through genetic engineering, obtaining a high-yielding lincomycin-producing strain, and providing technical support for increasing the fermentation yield of lincomycin in industrial production.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a method and application for increasing lincomycin production by modifying the SLCG_1608 gene of Streptomyces lincomyces. Background Technology

[0002] Streptomyces are an important source of secondary metabolites, producing compounds with various biological activities, such as antifungal, antiviral, antitumor, and antihypertensive compounds, the most important of which are antibiotics and immunosuppressants. With the rapid advancement of whole-genome sequencing technology, more and more Streptomyces genome sequences have been determined and analyzed, revealing that many of their biosynthetic gene clusters are in a silent state or have weak secondary product synthesis capabilities. Traditional physical or chemical mutagenesis techniques for obtaining high-yielding strains are time-consuming and uncertain, offering no theoretical guidance for breeding. The purpose of this invention is to obtain high-yielding lincomycin-producing strains through genetic engineering to directionally alter genes for the production of lincomycin or intermediate products.

[0003] Lincomycin, a lincosamide antibiotic primarily produced by *Streptomyces lincomyces*, and its derivative clindamycin are both clinically important antibiotics. They act on Gram-positive bacteria and some Gram-negative bacteria, targeting the peptidyl transferase center of the bacterial ribosome. By binding to the central loop of the 23S rRNA in the 50S subunit, they inhibit peptide chain elongation, thereby suppressing bacterial protein synthesis and affecting bacterial growth. Lincomycin is commonly used to treat bone and joint infections, and can also prevent postoperative intramural infections, oral infections, skin and mucous membrane infections. It is highly effective yet has low toxicity and is unlikely to develop cross-resistance, making it a promising antibiotic with broad application prospects.

[0004] Leucine-responsive regulatory proteins (Lrp) are widely found in bacteria and archaea and are important transcription factors that participate in regulating multiple cellular physiological processes, such as amino acid metabolism and transport, metabolism, DNA repair, and recombination. In recent years, several Lrp / AsnC family proteins, such as SACE_Lrp, SCO3361, SACE_5717, and SLCG_Lrp, have been discovered in actinomycetes and have been found to regulate the biosynthesis of various antibiotics. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method and application for improving the yield of lincomycin by modifying the SLCG_1608 gene of Streptomyces lincomyces.

[0006] The present invention solves the above-mentioned technical problems by adopting the following technical solutions:

[0007] A method for increasing lincomycin production by modifying the SLCG_1608 gene of Streptomyces lincomyces involves overexpressing the Lrp family transcriptional regulatory gene SLCG_1608 in Streptomyces lincomyces through genetic engineering to obtain a high-yielding strain of lincomycin, which is then used to produce lincomycin through fermentation. The nucleotide sequence of the SLCG_1608 gene is shown in SEQ ID NO.1.

[0008] As one of the preferred embodiments of the present invention, the amino acid sequence encoded by the SLCG_1608 gene is shown in SEQ ID NO.2.

[0009] As one of the preferred embodiments of the present invention, the SLCG_1608 gene product positively regulates lincomycin biosynthesis.

[0010] The above-mentioned method of increasing lincomycin yield by modifying the SLCG_1608 gene of Streptomyces lincomyces is applied to an industrial strain. The Lrp family regulatory gene SLCG_1608 is overexpressed in the industrial strain LA219X to obtain a high-yielding mutant strain for lincomycin production.

[0011] The advantages of this invention compared to the prior art are:

[0012] In this study, the positive regulator of lincomycin biosynthesis, SLCG_1608, was screened. By increasing the SLCG_1608 gene copy on the chromosome of Streptomyces lincomycin through genetic engineering, a high-yielding strain of lincomycin can be obtained, providing technical support for increasing the fermentation yield of lincomycin in industrial production.

[0013] Overexpression of the SLCG_1608 gene in *Streptomyces lincomyces* LCGL increased lincomycin production by 13.3%, while inactivation of SLCG_1608 resulted in a 37% decrease in lincomycin production, indicating that SLCG_1608 is a positive regulator of lincomycin biosynthesis. Using the high-yielding strain LA219X as the starting strain, overexpression of the SLCG_1608 gene on its chromosome increased lincomycin production by 16.4%, demonstrating that the technique of overexpressing SLCG_1608 to increase lincomycin production is also applicable to industrial strains. Attached Figure Description

[0014] Figure 1 This is a diagram showing the location of the SLCG_1608 gene and its surrounding genes on the chromosome.

[0015] Figure 2 This is a schematic diagram of the ΔSLCGL_1608 construction;

[0016] Figure 3This is a graph showing the PCR identification results of ΔSLCGL_1608 (in the graph, M: 5000bp DNA Marker; +: pKC1139-Δ1608; -: LCGL; 1: ΔSLCGL_1608);

[0017] Figure 4 This is a graph showing the PCR identification results of ΔSLCGL_1608 / pIB139-1608 complementation and LCGL / pIB139-1608 overexpression (in the graph, the PCR product is the Apr resistance gene, 776bp; M: 5000bp DNA Marker; +: pIB139 plasmid; -: ultrapure water; lane 1: ΔSLCGL_1608 / pIB139-1608 complementation strain; lane 2: LCGL / pIB139-1608 overexpression strain).

[0018] Figure 5 This is a graph showing the PCR identification results of the LA219X / pIB139-1608 strain (in the graph, M: 5000bp DNA Marker; +: pIB139 plasmid; -: ultrapure water; lane 1: LA219X / pIB139-1608 overexpressing strain).

[0019] Figure 6 The analysis of lincomycin production in the starting strains LCGL and ΔSLCGL_1608;

[0020] Figure 7 The effects of the SLCG_1608 gene on the morphological differentiation of the strain and the determination of the biomass of ΔSLCGL_1608 are shown in the figure. (Figure A shows the spore growth of LCGL and ΔSLCGL_1608 strains, with the left being LCGL strain and the right being ΔSLCGL_1608; Figure B shows the mycelial dry weight of LCGL and ΔSLCGL_1608 strains).

[0021] Figure 8 Analysis of lincomycin production in strains LCGL, ΔSLCGL_1608, ΔSLCGL_1608 / pIB139-1608, and LCGL / pIB139-1608;

[0022] Figure 9 This is an analysis of the transcriptional levels of genes related to the lincomycin biosynthesis gene cluster (in the figure, Figure A shows the transcriptional level analysis of regulatory factors; Figure B shows the transcriptional level analysis of resistance genes; and Figure C shows the transcriptional level analysis of structural genes).

[0023] Figure 10 Analysis of lincomycin yield in high-yielding lincomycin strains LA219X and LA219X / pIB139-1608. Detailed Implementation

[0024] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.

[0025] The strains and plasmids used in the following examples are shown in Table 1, and the synthesized primer sequences are shown in Table 2. Specifically, *Streptomyces lincosae* LCGL is an improved strain obtained based on *Streptomyces lincosae* LC-G (GenBank: CP022744.1), specifically by replacing the SLCG_7011 gene (GenBank: AXG58166.1) of *Streptomyces lincosae* LC-G with 4×attBΦC31; the 4×attBΦC31 is a 240bp sequence containing four attBΦC31 sites, as shown in SEQ ID NO. 3.

[0026] The *Escherichia coli* used in the following examples were cultured in liquid LB medium at 37°C or on solid LB plates supplemented with 1.25% agar. *Streptomyces lincosae* were cultured in tryptone soybean broth (TSBY) medium at 30°C or on modified Gauzes 1 (MGM) plates containing 1.8% agar.

[0027] PEG3350, lysozyme, TES, thiosphingolipids, and apramycin used in the following examples were purchased from Sigma-Aldrich. TSB, yeast extract, and peptone were purchased from Oxoid. Glycine, agar powder, sodium chloride, and other biological reagents were purchased from reagent companies. General handling techniques for *Escherichia coli* and *Streptomyces lincosae* were performed according to standard operating procedures. Primer synthesis and DNA sequencing were performed by Sangon Biotech (Shanghai) Co., Ltd.

[0028] Table 1. This invention relates to strains and plasmids.

[0029]

[0030] Table 2 This invention relates to primers.

[0031]

[0032]

[0033]

[0034] Example 1

[0035] Information related to the SLCG_1608 gene:

[0036] For the location of the SLCG_1608 gene and its neighboring genes on the chromosome, see [link to chromosome location information]. Figure 1.

[0037] According to the LCGL genome information, the SLCG_1608 gene is 498 bp in length and the protein monomer size is 18 kDa.

[0038] Specifically, the specific nucleotide sequence of the SLCG_1608 gene is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2.

[0039] Example 2

[0040] Construction of SLCG_1608 gene deletion mutant (see...) Figure 2 ):

[0041] Using the LCGL genome as a template, primers 1608-P1, 1608-P2, 1608-P3, and 1608-P4 were used to amplify 1.5 kb each of the upstream and downstream homologous arms of SLCG_1608, which were then recovered. At 37°C, the upstream and downstream homologous arms were digested with XbaI / EcoRI and XbaI / HindIII, respectively, and then recovered and quantified. Simultaneously, the pKC1139 plasmid was double-digested with EcoRI / HindIII, and the results were recovered and quantified. Based on the quantification results, the digested plasmid, upstream homologous arm, and downstream homologous arm were mixed at a ratio of 1:10:10 and ligated with T4 ligase at 22°C. The ligation products were then transformed into *E. coli* DH5α, diluted, and plated on Apr-resistant LB agar plates at 37°C for approximately 12 hours to grow single colonies. Finally, single clones were selected and cultured in LB liquid medium containing Apr resistance for expansion. They were then transferred to 5 mL of LB medium containing Apr resistance and cultured for 12 h. The pKC1139-Δ1608 plasmid was then extracted.

[0042] The obtained pKC1139-Δ1608 plasmid was transformed into LCGL protoplasts via PEG3350, and the SLCG_1608 knockout strain was constructed using homologous large fragment recombination technology. The specific experimental procedures were as follows: Approximately 4000 ng of pKC1139-Δ1608 was mixed with 50 μL of LCGL protoplasts, followed by the addition of 200 mL of PEG3350. After standing for 5 min, the mixture was diluted and spread onto R5 plates and incubated at 30℃ for approximately 20 h. Once membranous cells grew on the surface, Apr antibiotic was added for screening. After approximately 4 days, single colonies with blackened bases grew on the plates. These colonies were enriched on industrial plates containing Apr and incubated at 30℃ for approximately 3 days. Subsequently, the enriched spores were picked, diluted, and spread onto antibiotic-free R5 plates, and incubated at 37℃ for 3 days to induce plasmid loss. Finally, a portion of the monoclonal colonies from the R5 plates were plated onto Apr industrial plates, and the remaining monoclonal colonies were plated onto antibiotic-free industrial plates. Monoclonal colonies that could not grow on Apr plates but could grow on antibiotic-free plates were selected and cultured until spores were produced. A certain amount of spores were then scraped into sterile water and boiled at high temperature for 15 minutes. PCR identification was then performed using primers 1608-P5 and 1608-P6. Figure 3 The correct knockout strain was obtained.

[0043] Example 3

[0044] SLCG_1608 gene reversion and construction of overexpression strains:

[0045] Using the LCGL genome as a template, the SLCG_1608 gene fragment with NdeI and XbaI restriction sites at both ends was amplified and recovered using primers 1608-P7 and 1608-P8. The SLCG_1608 fragment and pIB139 plasmid were double-digested with NdeI and XbaI and recovered. At 22℃, T4 ligase was added to ligate the SLCG_1608 and pIB139 plasmids, which were then transformed into *E. coli* DH5α. The ligation was performed on LB agar plates containing Apr inhibitors, and single colonies grew at 37℃ for approximately 12 hours. Single colonies were selected and transferred to liquid LB medium for 5 hours of culture, followed by colony PCR identification. Correctly identified colonies were sequenced, and after successful sequencing, the pIB139-1608 plasmid was obtained through preservation.

[0046] The pIB139-1608 plasmid was introduced into LCGL and ΔSLCG_1608 protoplasts via protoplast transformation. Approximately 20 hours post-transformation, the culture medium was covered with apramycin aqueous solution and cultured until transformants appeared. Transformants were picked and plated onto industrial plates containing Apr resistance. After Apr-resistant transformants appeared, they were verified using primers Apr-F / R. Figure 4The correct strains ΔSLCGL_1608 / pIB139-1608 and LCGL / pIB139-1608 were obtained.

[0047] Example 4

[0048] The pIB139-1608 plasmid was transformed into LA219X protoplasts via PEG3350 to construct the LA219X / pIB139-1608 strain. The construction and screening methods were as described above, and the PCR identification results are as follows. Figure 5 .

[0049] Example 5

[0050] HPLC detection of Streptomyces lincosum fermentation products:

[0051] After culturing *Streptomyces lincosum* on slant agar for 7 days, 1 cm of... 2 Spore blocks were inoculated into seed culture medium and cultured at 30℃ and 240 rpm for 48 h with shaking. Then, they were transferred to fermentation medium and cultured at 30℃ and 240 rpm for 7 days with shaking. Then, 2 mL of bacterial culture was centrifuged at 12000 rpm for 10 min. 200 μL of supernatant was then mixed with 800 μL of anhydrous ethanol and centrifuged at 12000 rpm for 10 min. Finally, the supernatant was filtered through an organic filter membrane and injected into a test bottle for yield detection.

[0052] Example 6

[0053] Detection of Streptomyces lincosum mycelial biomass:

[0054] LCGL and ΔSLCGL_1608 were inoculated into 5 mL of liquid TSBY at the same inoculum size and cultured on a shaker at 30 °C for 48 h. Then, equal volumes of fresh LCGL and ΔSLCGL_1608 culture were transferred to YMG medium and cultured at 30 °C and 240 rpm for 7 days. During this period, 1 mL of fresh culture was collected every 24 h and stored at -20 °C. After fermentation, the 7-day sample was centrifuged at 12000 rpm for 10 min, and the supernatant was discarded. The cells were washed with 1 mL of anhydrous ethanol, centrifuged at 12000 rpm for 10 min, and the supernatant was discarded. The moistened cells were placed in a 65 °C oven for 2 days and weighed, and the cell mass was recorded. A biomass curve was plotted based on the dry weight of LCGL and ΔSLCGL_1608 cells according to the growth time.

[0055] Example 7

[0056] Transcriptional analysis of related genes in ΔSLCG_1608:

[0057] LCGL and ΔSLCG_1608 bacterial cultures were collected after 24 hours. The required RNA was obtained using a full-length gold RNA extraction kit, reversed into cDNA, and then detected using a real-time quantitative PCR instrument.

[0058] Example 8

[0059] This embodiment analyzes the results of one of the above embodiments:

[0060] 1. SLCG_1608 positively regulates the biosynthesis of lincomycin.

[0061] After fermentation of ΔSLCGL_1608 in fermentation medium for 168 h, lincomycin yield was analyzed by HPLC. The results showed that the yield of ΔSLCGL_1608 was approximately 37% lower than that of LCGL. Figure 6 ).

[0062] The results in summary confirm that SLCG_1608 is a positive regulatory gene for lincomycin biosynthesis in Streptomyces lincomyces, and that inactivating the SLCG_1608 gene in Streptomyces lincomyces through genetic engineering can reduce lincomycin production.

[0063] 2. Effects of SLCG_1608 gene deletion on spore morphology differentiation and cell growth

[0064] To further determine whether the SLCG_1608 gene regulates sporulation, LCGL and ΔSLCGL_1608 were simultaneously plated on MGM plates and incubated at 30°C for 7 days, with daily observation of sporulation growth. Results showed that there was no significant difference in sporulation morphology between LCGL and ΔSLCGL_1608. Figure 7 A) indicates that the deletion of the SLCG_1608 gene does not affect spore formation.

[0065] The cell dry weights of LCGL and ΔSLCGL_1608 were measured, and corresponding change curves were plotted. The results showed that the biomass of ΔSLCGL_1608 was not significantly different from that of LCGL. Figure 7 B) suggests that the deletion of the SLCG_1608 gene did not affect the primary metabolism of the bacteria.

[0066] 3. SLCG_1608 gene reversion and overexpression

[0067] To verify that the decrease in lincomycin yield in ΔSLCGL_1608 was caused by the deletion of the SLCG_1608 gene, the SLCG_1608 gene expression vector pIB139-1608 was introduced into protoplasts of LCGL and ΔSLCGL_1608, respectively. The revertant strain ΔSLCGL_1608 / pIB139-1608 and the overexpression strain LCGL / pIB139-1608 were identified by PCR. Shake-flask fermentation was performed on the LCGL and ΔSLCGL_1608 series strains. HPLC results showed that the lincomycin yield in ΔSLCGL_1608 decreased by 37% compared to LCGL; the lincomycin yield in the revertant strain ΔSLCGL_1608 / pIB139-1608 recovered to the level of lincomycin in LCGL; and the lincomycin yield in LCGL / pIB139-1608 increased by 13.3% compared to LCGL. Figure 8 ).

[0068] The above results indicate that SLCG_1608 positively regulates the biosynthesis of lincomycin.

[0069] 4. Transcriptional analysis of related genes in ΔSLCG_1608

[0070] qRT-PCR data showed that, compared with LCGL, the transcriptional levels of regulatory genes, resistance genes, and most structural genes within the lincomycin biosynthesis gene cluster in ΔSLCGL_1608 were downregulated to varying degrees. Figure 9 This indicates that SLCG_1608 positively regulates the transcriptional level of genes within the cluster, thereby controlling the biosynthesis of lincomycin.

[0071] 5. Overexpression of the SLCG_1608 gene in the high-yielding strain LA219X can increase lincomycin production.

[0072] The LA219X / pIB139-1608 strain and the high-yielding strain LA219X were plate-coated for activation, and then inoculated into shake flasks containing industrial seed culture medium. After 48 h of incubation at 30℃ and 240 rpm, they were transferred to fermentation medium and cultured for another 168 h. After fermentation, extraction and concentration were performed, and HPLC analysis showed that compared to LA219X, the LA219X / pIB139-1608 strain exhibited a 16.4% increase in lincomycin yield. Figure 10 This indicates that the SLCG_1608 gene in the high-yielding strain LA219X is also involved in controlling lincomycin production.

[0073] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for modifying Streptomyces lincosum SLCG_1608 A method for increasing lincomycin production through gene therapy, characterized in that, Lrp family transcriptional regulatory genes were overexpressed in Streptomyces lincosae LA219X via genetic engineering. SLCG_1608 A high-yield strain of lincomycin was obtained, and the obtained strain was used to produce lincomycin through fermentation; among which, SLCG_1608 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The method of modifying Streptomyces lincosum according to claim 1 SLCG_1608 A method for increasing lincomycin production through gene therapy, characterized in that, The SLCG_1608 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

2.

3. The method of modifying Streptomyces lincosum according to claim 1 SLCG_1608 A method for increasing lincomycin production through gene therapy, characterized in that, The SLCG_1608 Gene products positively regulate lincomycin biosynthesis.

4. A kind SLCG_1608 The application of genetically modified Streptomyces lincosinate is characterized by, The method constructed using any one of claims 1 to 3 SLCG_1608 Genetically modified Streptomyces lincomycin is used for the fermentation production of lincomycin.

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