Method and application of increasing lincomycin production by modifying the gene of Streptomyces lincomycin SLCG_2185

Abstract

The present invention provides a method for increasing the yield of lincomycin by modifying the lincomycin Streptomyces SLCG_2185 gene, overexpressing the FadR family transcriptional regulatory gene SLCG_2185 in the lincomycin Streptomyces through genetic engineering, obtaining a lincomycin high-yield engineered strain, and fermenting and producing lincomycin with the obtained strain; the nucleotide sequence of the SLCG_2185 gene is shown in SEQ ID NO.1. The present invention also provides an application of the method for increasing the yield of lincomycin by modifying the lincomycin Streptomyces SLCG_2185 gene. The present invention screens the lincomycin biosynthesis positive regulator SLCG_2185, and overexpresses the SLCG_2185 gene copy on the lincomycin chromosome through genetic engineering, so as to obtain a lincomycin high-yield strain, thereby providing technical support for increasing the fermentation yield of lincomycin in industrial production.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to a method and application of increasing the yield of lincomycin by transforming the gene of Streptomyces lincomycin SLCG_2185. Background Art

[0002] Actinomycetes are the most important producers of antibiotics. Currently, more than 20,000 microbial bioactive substances have been found to be derived from actinomycetes, including more than 13,800 natural products with antibiotic activity. In addition to the widely used medical or agricultural antibiotics such as streptomycin, kanamycin, spinosad, lincomycin, etc., they also include immunomodulators, receptor antagonists, etc.

[0003] In recent years, with the rapid development of genome sequencing technology, a large number of Streptomyces genome sequences have been measured and analyzed. In addition to finding antibiotics of known structure, a large number of secondary metabolite biosynthetic gene clusters are also contained in the Streptomyces genome. However, these gene clusters are either silent or have weak biosynthetic capabilities, and high-yield strains required for industrial production need to be obtained by screening. In the past, industrial production strains were mainly obtained by physical or chemical mutagenesis methods, but traditional mutagenesis techniques are not only time-consuming, but also have large randomness and cannot provide theoretical guidance for breeding. The purpose of the present invention is to obtain lincomycin high-yield strains by genetic engineering approach directed gene modification, which is used for lincomycin or intermediate production.

[0004] In 1962, Marson et al. isolated the first strain of Streptomyces lincolnensis NRRL2936 from soil near Lincoln, Nebraska, USA. This strain produces a lincosamide antibiotic, lincomycin A (Lin-A), which has strong effects on Gram-positive bacteria both in vivo and in vitro, and also has a certain inhibitory effect on Gram-negative bacteria. At present, lincomycin is widely used in veterinary drugs and raw materials. Its chemical derivatives, such as clindamycin, have great medicinal value and are one of the few drugs used clinically to treat Gram-negative anaerobic bacteria. With the growing demand for lincomycin and its derivatives in medicine and commerce, it is of great economic value to construct high-yield industrial strains to increase their yield. At present, the functions of most genes in the lincomycin biosynthetic gene cluster have been analyzed, and the lincomycin biosynthetic pathway has been very clear, which also lays an important foundation for the high-quality breeding of lincomycin. Although there have been some reports on the high production of lincomycin through genetic engineering of Streptomyces lincomycin, the research on the regulation of lincomycin biosynthesis is still relatively limited, which restricts the improvement of yield through genetic regulation of Streptomyces lincomycin.

[0005] GntR family transcriptional regulators are widely distributed in bacteria. They can regulate the expression of different genes according to the type and amount of available nutrients in the environment to activate or inhibit a variety of primary metabolic pathways, and further participate in the interconnection of primary and secondary metabolic pathways. According to the diversity of the C-terminal ligand binding domain of the GntR family, it can be divided into seven subfamilies, including FadR, HutC, MocR, ytrA, AraR, Deva and PLMA. Among them, the FadR subfamily has the most members, accounting for about 40%, and is the main transcriptional regulator of fatty acid balance in prokaryotes. Most FadRs can also act on the intersection of various metabolic pathways, such as the synthesis pathways of aspartate, pyruvate, lactate, malonate and gluconic acid. The important transcriptional regulators of primary metabolism of Streptomyces are closely related to strain morphological differentiation and antibiotic biosynthesis. Studies have shown that the FadR family regulator WhiH in Streptomyces coelicolor can regulate the separation and spore formation of aerial hyphae; SCO1678 can regulate the biosynthesis of actinomycetophthrin. This suggests that the FadR family may also play an important role in the biosynthesis of other Streptomyces antibiotics, but there are few reports on the research of this family, especially in Streptomyces lincomycetes, there has been no relevant research on FadR family regulatory factors. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a method and application for increasing the yield of lincomycin by transforming the gene of Streptomyces lincomycin SLCG_2185.

[0007] The present invention adopts the following technical solutions to solve the above technical problems:

[0008] A method for improving the yield of lincomycin by modifying the lincomycin Streptomyces lincomycin SLCG_2185 gene, overexpressing the FadR family transcriptional regulatory gene SLCG_2185 in the lincomycin Streptomyces lincomycin through genetic engineering to obtain a lincomycin high-yield engineered strain, and using the obtained strain to ferment and produce lincomycin; wherein the nucleotide sequence of the SLCG_2185 gene is shown in SEQ ID NO.1.

[0009] As one of the preferred embodiments of the present invention, the amino acid sequence encoded by the SLCG_2185 gene is shown in SEQ ID NO.2.

[0010] As one of the preferred embodiments of the present invention, the SLCG_2185 gene product positively regulates lincomycin biosynthesis.

[0011] An application of the above method for increasing lincomycin production by modifying the lincomycin Streptomyces SLCG_2185 gene: overexpressing the FadR family regulatory gene SLCG_2185 in the lincomycin industrial high-yield strain LA219X to obtain a high-yield mutant for lincomycin production.

[0012] The advantages of the present invention compared to the prior art are:

[0013] In the present invention, a positive regulator of lincomycin biosynthesis SLCG_2185 was screened out, and a lincomycin high-yield strain was obtained by overexpressing a copy of the SLCG_2185 gene on the chromosome of Streptomyces lincomycin through genetic engineering, providing technical support for increasing the fermentation yield of lincomycin in industrial production.

[0014] When the SLCG_2185 gene was overexpressed in Streptomyces lincomycin LCGL, the lincomycin yield increased by 14.6%, while the lincomycin yield of the ΔSLCG_2185 deletion mutant decreased by 20.2%, indicating that SLCG_2185 is a positive regulatory factor involved in the biosynthesis of lincomycin. At the same time, the industrial high-yield strain LA219X in the field was used as the starting strain, and the SLCG_2185 gene was overexpressed on its chromosome, which increased the lincomycin yield by 17.8%, indicating that the technology of overexpressing the SLCG_2185 gene to increase the lincomycin yield is also applicable to industrial high-yield strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is the location information of SLCG_2185 gene and its neighboring genes on chromosome;

[0016] Figure 2 is a schematic diagram of the construction of the ΔSLCG_2185 mutant;

[0017] Figure 3 This is the PCR identification result of the ΔSLCG_2185 mutant ( Figure 3 Middle, M: 5000 bp DNA Marker; +: pKC1139-Δ2185; -: LCGL; 1: ΔSLCGL_2185 strain);

[0018] Figure 4 PCR identification diagram of ΔSLCGL_2185 / pIB139 empty vector, ΔSLCGL_2185 / pIB139-2185 complementation, LCGL / pIB139 empty vector strain, and LCGL / pIB139-2185 overexpression ( Figure 4 In the figure, the PCR product is apr resistance gene (776 bp); M: 5000 bp DNA Marker; -: ultrapure water +: pIB139 plasmid; Lane 1: ΔSLCGL_2185 / pIB139 complemented empty control strain; Lane 2: ΔSLCGL_2185 / pIB139-2185 complemented strain; Lane 3: LCGL / pIB139 overexpression empty control strain; Lane 4: LCGL / pIB139-2185 overexpression strain);

[0019] Figure 5 This is the PCR identification diagram of the LA219X / pIB139-2185 strain ( Figure 5 Middle, M: 5000 bp DNA Marker; +: pIB139 plasmid; Lanes 1, 2: LA219X / pIB139-2185 overexpression strain);

[0020] Figure 6 It is the analysis diagram of lincomycin production of the starting strain LCGL and the deletion mutant strain ΔSLCGL_2185;

[0021] Figure 7 This is a diagram of the transcription level analysis of genes related to the lincomycin biosynthesis gene cluster ( Figure 7 Figure 1 shows the transcriptional analysis of regulatory factors; Figure 1 shows the transcriptional analysis of resistance genes; Figure 1 shows the transcriptional analysis of structural genes);

[0022] Figure 8 The effect of SLCG_2185 gene on strain morphological differentiation and the determination of biomass of ΔSLCGL_2185 mutant strain ( Figure 8 Figure A shows the spore growth of the ΔSLCGL_2185 mutant and the starting strain LCGL, and the left: ΔSLCGL_2185 mutant, right: LCGL strain; Figure B shows the dry weight of mycelium of the ΔSLCGL_2185 mutant and the starting strain LCGL strain);

[0023] Fig. 9 is a graph analyzing lincomycin production of LCGL, ΔSLCGL_2185, ΔSLCGL_2185 / pIB139, ΔSLCGL_2185 / pIB139-2185, LCGL / pIB139, and LCGL / pIB139-2185 strains;

[0024] Fig.10 This is an analysis of lincomycin production in the industrial high-yield lincomycin strains LA219X and LA219X / pIB139-2185. DETAILED DESCRIPTION

[0025] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0026] The strains and plasmids used in the following examples are shown in Table 1, and the synthesized primer sequences are shown in Table 2. The starting strain, Streptomyces lincone LCGL, is an improved strain obtained on the basis of Streptomyces lincone LC-G (GenBank: CP022744.1), specifically: the SLCG_7011 gene (GenBank: AXG58166.1) of Streptomyces lincone LC-G is replaced with 4×attBΦC31; the 4×attBΦC31 is a 240 bp base sequence containing 4 attBΦC31 site sequences, and the base sequence is shown in SEQ ID NO.3.

[0027] The E. 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. The lincomycin-producing bacteria Streptomyces lincomycin were cultured in tryptone soy broth (TSBY) medium at 30°C or on modified Gould's 1 (MGM) plates containing 1.8% agar.

[0028] PEG3350, lysozyme, TES, casamino acid, thiostrepton, and apramycin used in the following examples were purchased from Sigma. TSB, yeast extract, and peptone were purchased from Oxoid. Glycine, agar powder, sodium chloride, and other biological reagents were purchased from a reagent company. General operation techniques for Escherichia coli and Streptomyces lincomycin were performed according to standard procedures. Primer synthesis and DNA sequencing were performed by Sangon Biotech (Shanghai) Co., Ltd.

[0029] Table 1 The present invention relates to bacterial species, plasmids and their main properties

[0030]

[0031]

[0032] Table 2 The present invention relates to primers

[0033]

[0034]

[0035]

[0036]

[0037] Example 1

[0038] SLCG_2185 gene related information:

[0039] The location of SLCG_2185 and adjacent genes on the chromosome of Streptomyces lincolnii can be found in Figure 1 .

[0040] According to the LCGL genome information, the length of the SLCG_2185 gene is 942 bp, and the protein monomer size is 33.6 KDa. Specifically, the nucleotide sequence of the SLCG_2185 gene is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2.

[0041] Example 2

[0042] Construction of SLCG_2185 gene deletion mutant (see Figure 2 ):

[0043] Using LCGL as a template, 2185-P1, 2185-P2, 2185-P3, and 2185-P4 as primers, the upstream and downstream homology arms of SLCG_2185 were amplified 1.8 kb each, and the gel recovery kit was used to recover them. The upstream and downstream homology arms were digested with XbaI / EcoRI and XbaI / HindIII, respectively, and the fragments after digestion were recovered by gel and the DNA was quantified by UV spectrophotometer. At the same time, the pKC1139 plasmid was double digested with EcoRI / HindIII and then recovered and quantified. According to the quantitative results, the digested plasmid, upstream homology arm, and downstream homology arm were mixed in a ratio of 1:5:5, T4 ligase was added, and the water bath connection process was carried out at 22°C. Then, all the ligation products were transformed into Escherichia coli DH5α, and then coated on LB plates containing 0.1% Apr and cultured for about 20 hours to grow a single clone. Finally, a single clone was selected and cultured in liquid culture medium for expansion, and the plasmid was extracted for double enzyme digestion verification to obtain a DH5α-positive strain carrying the plasmid pKC1139-Δ2185.

[0044] The obtained pKC1139-Δ2185 plasmid was transformed into the protoplasts of LCGL by PEG3350, and the construction of the SLCG_2185 knockout strain was completed by homologous large fragment recombination technology. The specific experimental operation is as follows: about 5000-8000ng of pKC1139-Δ2185 was mixed with LCGL protoplasts and PEG3350 was added. The mixture was spread on the R5 plate at 30℃ and cultured for about 20h. After the film-like bacteria grew on the surface, Apr antibiotics were added for coverage screening. After 3-5 days, the single clone grown on the plate and blackened at the bottom was the single exchange strain, which was enriched on the industrial plate containing Apr and cultured at 30℃ for 3-5 days. Subsequently, the enriched spores were scraped and diluted and spread on the R5 plate without resistance, and cultured at 37℃ for 5-6 days to lose the plasmid. Finally, half of the monoclonal clones on the R5 plate were spread on the Apr industrial plate and the other half on the antibiotic-free industrial plate. The monoclonal clones that did not grow on the Apr plate but grew on the antibiotic-free plate were selected for enrichment. After the monoclonal clones produced spores, some spores were scraped as templates and 2185-P5 and 2185-P6 were used as primers for PCR identification. Figure 3 , and obtain the correct knockout strain.

[0045] Example 3

[0046] Construction of SLCG_2185 gene restoration and overexpression strains:

[0047] Using LCGL as a template and 2185-P7 and 2185-P8 as primers, the SLCG_2185 gene with NdeI and XbaI restriction sites at both ends was amplified. The amplified and recovered SLCG_2185 fragment and pIB139 plasmid were treated with NdeI and XbaI double restriction enzymes. After recovery, the SLCG_2185 and pIB139 plasmids were connected and transformed into Escherichia coli DH5α, and spread on LB plates with Apr resistance. At 37°C, a single clone will grow in about 16 hours. The single clone was transferred to liquid LB culture medium and cultured for 5-8 hours before PCR identification of the bacterial liquid. The correctly identified bacteria were sent for sequencing, and after sequencing was correct, the pIB139-2185 plasmid was obtained by seed preservation.

[0048] The obtained pIB139-2185 plasmid was introduced into the protoplasts of the starting strains LCGL and ΔSLCG_2185 by protoplast transformation. The pIB139 empty plasmid was transferred into the LCGL protoplasts in the same way. About 24 hours after transformation, the culture medium was covered with an aqueous solution of apramycin and cultured until transformants grew out. The transformants were selected and spread on industrial plates with Apr resistance. After the Apr-resistant transformants grew out, PCR identification was performed using primers Apr-F / R. Figure 4, and obtained the correct ΔSLCGL_2185 / pIB139, ΔSLCGL_2185 / pIB139-2185, LCGL / pIB139, and LCGL / pIB139-2185 strains.

[0049] Example 4

[0050] Construction of LA219X / pIB139-2185 lincomycin high-yield strain among industrial high-yield strains:

[0051] The pIB139-2185 plasmid was transformed into the protoplasts of LA219X by PEG3350 to construct the LA219X / pIB139-2185 strain. The construction and screening method was as described above. The PCR identification results were as follows: Figure 5 .

[0052] Example 5

[0053] HPLC detection of fermentation products of Streptomyces lincomyces:

[0054] After culturing Streptomyces lincosus on the slant medium for 7 days, dig a 1 cm 2 Inoculate in seed culture medium, shake culture at 30°C, 240rpm for 48 hours, transfer to fermentation medium, shake culture at 30°C, 240rpm for 7 days, then take 2mL of bacterial solution and centrifuge at 12000rpm for 10min; then take 200μl of supernatant and add 800μl of ethanol to mix, centrifuge at 12000rpm for 10 minutes; finally, inject the supernatant into the detection bottle through an organic filter membrane for yield detection.

[0055] Example 6

[0056] Streptomyces lincomyces mycelium biomass detection:

[0057] The ΔSLCGL_2185 mutant and LCGL were inoculated into 30 mL of liquid TSBY with the same inoculation amount. After culturing at 30 °C for 48 hours, an equal amount of LCGL and ΔSLCGL_2185 fresh bacterial liquid was taken into YMG medium and cultured at 30 °C and 240 rpm for 7 days. During this period, 1 mL of bacterial liquid was taken out every 24 hours and stored in a -80 °C refrigerator. After the fermentation was completed, the seven-day sample was centrifuged at 12000 rpm for 10 minutes, and the supernatant was discarded; 1 mL of anhydrous ethanol was added to wash the cells, and the cells were centrifuged at 12000 rpm for 10 minutes, and the supernatant was discarded; the wet cells were placed in a 65 °C oven for 2 days and weighed, and the cell mass was recorded. The cell dry weight of ΔSLCGL_2185 and LCGL was plotted against the growth time to draw the cell biomass curve.

[0058] Example 7

[0059] Transcription analysis of related genes in ΔSLCG_2185:

[0060] The bacterial culture fluid of ΔSLCG_2185 and wild strain LCGL was collected for 24 hours, and the required RNA was obtained using the Total Gold RNA Extraction Kit. After being reversed into cDNA, it was detected using a real-time fluorescence quantitative PCR instrument.

[0061] Example 8

[0062] The results of the above-mentioned embodiments of this embodiment are analyzed as follows:

[0063] 1. SLCG_2185 positively regulates the biosynthesis of lincomycin

[0064] After 168 h of fermentation in the fermentation medium, the lincomycin production of ΔSLCGL_2185 was detected by HPLC, and it was found that the production of lincomycin in ΔSLCGL_2185 was 20.2% lower than that of the original strain LCGL ( Figure 6 ). qRT-PCR data showed that compared with the starting strain LCGL, the transcription levels of regulatory genes, resistance genes and most structural genes in the lincomycin biosynthesis gene cluster in the ΔSLCGL_2185 mutant were downregulated to varying degrees ( Figure 7 ), indicating that SLCG_2185 positively regulates the transcription level of genes in the cluster and thus controls the biosynthesis of lincomycin.

[0065] 2. Effect of SLCG_2185 gene deletion on spore morphological differentiation and bacterial growth

[0066] In order to determine whether the SLCG_2185 gene regulates the spore formation of bacteria, the mutant strain ΔSLCGL_2185 and the control strain LCGL were simultaneously spread on MGM plates and cultured at 30°C for 48h and 72h to observe the spore growth of the strains. The results showed that compared with LCGL, the spore morphology of the ΔSLCGL_2185 mutant strain was not significantly different ( Figure 8 A), indicating that the deletion of the SLCG_2185 gene does not affect spore formation. The dry weight of the ΔSLCGL_2185 mutant and the LCGL strain after fermentation for 168 h was measured, and the corresponding change curve was drawn. The results showed that the biomass of ΔSLCGL_2185 was not much different from that of LCGL ( Figure 8 B), suggesting that the deletion of the SLCG_2185 gene did not affect the primary metabolism of the bacteria.

[0067] 3. SLCG_2185 gene restoration and overexpression

[0068] In order to further verify that the decrease in lincomycin production in the mutant ΔSLCGL_2185 was caused by the deletion of the SLCGL_2185 gene, the SLCG_2185 gene expression vector pIB139-2185 and pIB139 vector (control) were introduced into the protoplasts of the ΔSLCGL_2185 mutant and LCGL, respectively, to obtain the restored strain ΔSLCGL_2185 / pIB139-2185 and the empty vector ΔSLCGL_2185 / pIB139, the overexpression strain LCGL / pIB139-2185 and the empty vector LCGL / pIB139. The LCGL and ΔSLCGL_2185 strains were fermented in shake flasks. The results of HPLC showed that the lincomycin production in ΔSLCGL_2185 was 20.2% lower than that in LCGL; the lincomycin production in the reverted strain ΔSLCGL_2185 / pIB139-2185 was restored to the level of lincomycin in LCGL; the lincomycin production in LCGL / pIB139-2185 was 14.6% higher than that in LCGL ( Fig. 9 These results indicate that SLCG_2185 positively regulates the biosynthesis of lincomycin and overexpression of SLCG_2185 in LCGL effectively increases the production of lincomycin.

[0069] 4. The industrial strain LA219X / pIB139-2185 overexpressing the SLCG_2185 gene can increase the yield of lincomycin

[0070] The LA219X / pIB139-2185 strain and the industrial strain LA219X were plated and activated, and then inoculated into shake flasks of industrial seed culture medium, respectively, and cultured at 30°C and 240rpm for 48 hours, and then transferred to fermentation medium and continued to be cultured for 168 hours. After the fermentation, the extraction and concentration were analyzed by HPLC. Compared with the starting strain LA219X, the lincomycin yield of LA219X / pIB139-2185 increased by 17.8% ( Fig.10 ), which indicates that the SLCG_2185 gene in the high-yield strain LA219 is also involved in regulating the production of lincomycin.

[0071] 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 in the protection scope of the present invention.

Claims

1. A method for modifying Streptomyces lincomyces SLCG_2185 A method for genetically increasing lincomycin production, characterized in that: Overexpression of FadR family transcriptional regulatory genes in Streptomyces lincomyces LA219X through genetic engineering SLCG_2185 , obtaining a lincomycin high-yield engineered strain, and using the obtained strain to ferment and produce lincomycin; wherein, the SLCG_2185 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The method according to claim 1, wherein the modified Streptomyces lincomyces SLCG_2185 A method for genetically increasing lincomycin production, characterized in that: Said SLCG_2185 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

2.

3. The method according to claim 1 for modifying Streptomyces lincomyces SLCG_2185 A method for genetically increasing lincomycin production, characterized in that: Said SLCG_2185 The gene product positively regulates lincomycin biosynthesis.

4. A SLCG_2185 The application of genetically modified Streptomyces lincomyces is characterized by: The method obtained by any one of claims 1 to 3 SLCG_2185 Genetically modified Streptomyces lincomycin is used for the fermentation production of lincomycin.

Images