Methods and applications for improving erythromycin yield by modifying the SACE_5680 gene in *Rhodotorula polyspora*.

Abstract

This invention relates to the field of genetic engineering technology and provides a method for increasing erythromycin yield by modifying the SACE_5680 gene of *Rhodotorula sacchariformis*. The method involves deleting the GntR family SACE_5680 gene in *Rhodotorula sacchariformis* through genetic engineering, thereby obtaining a high-yielding engineered strain of *Rhodotorula sacchariformis* for erythromycin production. Erythromycin is then produced by fermentation using this high-yielding engineered strain. The nucleotide sequence of the SACE_5680 gene is shown in SEQ ID NO. 1. This invention also provides an application of the above method. In this study, the negative regulator of erythromycin biosynthesis, SACE_5680, was screened. By deleting a copy of the SACE_5680 gene on the chromosome of *Rhodotorula sacchariformis* through genetic engineering, a high-yielding strain of erythromycin can be obtained, providing technical support for increasing erythromycin fermentation yield 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 erythromycin production by modifying the SACE_5680 gene of *Rhodotorula sacchariformis*. Background Technology

[0002] Erythromycin is produced by the secondary metabolism of *Rhodotorula sacchariformis* and belongs to the typical polyketide class of antibiotics. Its components include erythromycin A (Er-A), erythromycin B (Er-B), erythromycin C (Er-C), erythromycin D (Er-D), erythromycin E (Er-E), and erythromycin F (Er-F). This class of antibiotics has broad-spectrum antibacterial activity, similar to that of penicillin, and exhibits strong inhibitory activity against Gram-positive bacteria. Among the various components of erythromycin, erythromycin A is the most widely used clinically due to its highest antibacterial activity. Erythromycin derivatives (clarithromycin, azithromycin, roxithromycin, telithromycin, etc.) are also widely used to treat infectious diseases. The annual sales of erythromycin and its derivatives reach tens of billions of dollars.

[0003] Erythromycin plays a vital role in the pharmaceutical field, but its yield still needs improvement. Traditional methods for optimizing fermentation conditions to increase erythromycin A production are time-consuming and uneconomical, making them unsuitable for widespread application. However, using genetic engineering to increase the copy number of the synthetic gene in the chromosome of *Rhodotorula sacchari*, or modifying the regulatory gene through gene knockout, to obtain high-yielding erythromycin strains shows great promise.

[0004] Ramos et al. classified prokaryotic transcription regulators into 16 families based on sequence similarity, structure, and function: LysR, AraC / XylS, TetR, LuxR, LacI, ArsR, IcIR, MerR, AsnC, MarR, NtrC (EBP), OmpR, DeoR, Coldshock, GntR, and Crp. The GntR family of transcription regulators is the most widely distributed helix-turn-helix (HTH) transcription regulator in bacteria. This family of transcription regulators contains two functional domains: an N-terminal DNA-binding domain and a C-terminal effector-binding / oligomerization domain. While the amino acid sequence of the DNA domain is highly conserved among GntR family members, the amino acid sequences of the effector-binding / oligomerization domain exhibit significant differences. Many GntR family transcription factors have been identified, regulating various bacterial cellular processes such as motility, glucose metabolism, antibiotic resistance, and pathogenicity. However, there is currently very little research on GntR family regulatory genes in the secondary metabolism of *Rhodotorula sacchariformis*. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a method and application for increasing erythromycin production by modifying the SACE_5680 gene of *Rhodotorula sacchariformis*.

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

[0007] A method for increasing erythromycin yield by modifying the SACE_5680 gene of *Rhodotorula sacchariformis* involves deleting the GntR family SACE_5680 gene in *Rhodotorula sacchariformis* using genetic engineering methods, thereby obtaining a high-yield engineered strain of *Rhodotorula sacchariformis* for erythromycin production. Erythromycin is then produced by fermentation using this high-yield engineered strain. The nucleotide sequence of the SACE_5680 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 SACE_5680 gene is shown in SEQ ID NO.2.

[0009] As one of the preferred embodiments of the present invention, the SACE_5680 gene product negatively regulates erythromycin biosynthesis.

[0010] An application of the above-mentioned method for increasing erythromycin yield by modifying the SACE_5680 gene of *Rhodotorula sacchari* involves knocking out the SACE_5680 gene in a high-yield industrial strain to obtain a high-yield mutant strain for use in erythromycin production.

[0011] As one of the preferred embodiments of the present invention, the industrial high-yield strain is specifically selected as the industrial strain WB of Rhodotorula polyspora, and correspondingly, the high-yield mutant strain is the WB-△SACE_5680 strain.

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

[0013] This invention screened out the negative regulator of erythromycin biosynthesis, SACE_5680. By deleting a copy of the SACE_5680 gene from the chromosome of *Rhodotorula sacchariflora* through genetic engineering, a high-yielding erythromycin-producing strain was obtained, providing technical support for increasing erythromycin fermentation yield in industrial production. Specifically, knocking out the SACE_5680 gene in *Rhodotorula sacchariflora* A226 increased erythromycin yield by 22%, while reintroducing the SACE_5680 gene into the ΔSACE_5680 deletion mutant restored erythromycin yield, indicating that SACE_5680 is a negative regulator involved in erythromycin biosynthesis. Furthermore, using the high-yielding industrial strain WB as the starting strain, deleting the SACE_5680 gene from its chromosome increased erythromycin yield by 53%, demonstrating that the technique of increasing erythromycin yield by deleting the SACE_5680 gene is also applicable to high-yielding industrial strains. Attached Figure Description

[0014] Figure 1 This is a diagram showing the location of the SACE_5680 gene and its neighboring genes on the chromosome.

[0015] Figure 2 This is a schematic diagram of the construction of the △SACE_5680 mutant strain;

[0016] Figure 3 This is a PCR identification diagram of the △SACE_5680 mutant strain (in the diagram, the SACE_5680 gene is not knocked out, 666bp; the SACE_5680 gene is knocked out, 360bp; M, 5000bp DNA Marker).

[0017] Figure 4 This is a PCR identification diagram of the ΔSACE_5680 / pIB139-5680 complement strain (in the diagram, the PCR product is the apramycin resistance gene, 776bp; M, 5000bp DNA Marker);

[0018] Figure 5 The analysis of erythromycin A production in the starting strain A226 and the deletion mutant strain ΔSACE_5680 (in the figure, "***" indicates a significant difference, p<0.001);

[0019] Figure 6 The HPLC analysis of erythromycin A in the starting strain A226, the deletion mutant ΔSACE_5680, the deletion complementation strain ΔSACE_5680 / pIB139-5680, and the complementation empty vector control strain ΔSACE_5680 / pIB139 is shown in the figure ("***" indicates significant difference, p<0.001).

[0020] Figure 7 This is a graph showing the biomass measurement results of mycelium of the ΔSACE_5680 mutant strain and the wild-type A226 strain.

[0021] Figure 8 The transcriptional levels of genes related to the erythromycin synthesis gene cluster in ΔSACE_5680 are shown in the figure ("**" indicates p<0.01, "***" indicates p<0.001).

[0022] Figure 9 The HPLC analysis of erythromycin A yield of high-yielding strain WB and deletion mutant strain WB / ΔSACE_5680 is shown in the figure ("**" indicates significant difference, p<0.01). Detailed Implementation

[0023] 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.

[0024] 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 was *Rhodotorula sacchariformis* A226 (CGMCC 8279), a strain that can be purchased directly.

[0025] Meanwhile, 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. *Rhodotorula glycosides*, the erythromycin-producing bacterium, and its engineered strains were cultured in tryptone soybean broth (TSB) medium at 30°C or on R3M plates containing 2.2% agar.

[0026] In the following examples, PEG3350, lysozyme, TES, caseinate, thiosporin, and apramycin 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 procedures for Escherichia coli and Rhodotorula sacchariformis were performed according to standard operating procedures. Primer synthesis and DNA sequencing were performed by Sangon Biotech (Shanghai) Co., Ltd.

[0027] Table 1. The bacterial strains, plasmids, and their main properties involved in this invention.

[0028]

[0029]

[0030] Table 2 This invention relates to primers.

[0031]

[0032]

[0033]

[0034] Example 1

[0035] Analysis of the SACE_5680 gene:

[0036] The locations of SACE_5680 and neighboring genes on the chromosome of *Rhodotorula sacchari* are shown below. Figure 1 .

[0037] The nucleotide sequence of the SACE_5680 gene is shown in SEQ ID NO.1. The gene length is 666 bp, the encoded amino acid sequence is shown in SEQ ID NO.2, and the protein monomer size is 24.4 kDa.

[0038] Example 2

[0039] Construction of SACE_5680 gene deletion mutant strain (see...) Figure 2 ):

[0040] Using genomic DNA of *Rhodotorula polyspora* A226 as a template, 1.5 kb upstream and downstream homologous arms were amplified by PCR using primers 5680UF / 5680UR and 5680DF / 5680DR, respectively. The upstream and downstream homologous arms were digested with Hind III and Xba I and EcoRI and Xba I, and then recovered. Simultaneously, the vector pKC1139 was knocked out by Hind III and EcoRI and recovered. The recovered upstream and downstream homologous arms were ligated with the pKC1139 plasmid and transformed into DH5α. The pKC1139-Δ5680 was obtained by PCR and restriction enzyme digestion identification.

[0041] The constructed plasmid pKC1139-Δ5680 was transformed into the prepared *Rhodotorula sacchariformis* A226 protoplasts, then plated onto antibiotic-free R3M agar plates (after inverting and drying, placed in a 37°C oven for approximately 48 hours), and incubated at 30°C for about 20 hours. A final concentration of 50 μg / mL apramycin was added to cover the plates, and incubation continued at 30°C for 4–5 days until single colonies emerged. The emerged single colonies were then transferred to R3M agar plates containing 50 μg / mL apramycin for enrichment, and spores emerged after 4–5 days of incubation at 30°C. A small amount of spores was streaked onto antibiotic-free R3M agar plates and then incubated at 37°C for 2–3 days to complete plasmid relaxation and loss. When single colonies emerge from the plates, half of the spores are placed on antibiotic-free R3M solid plates, and the other half on R3M solid plates containing 50 μg / mL apramycin. Incubate at 30°C for 4–5 days. For the strains that do not grow on the apramycin-containing R3M solid plates but do grow on antibiotic-free R3M solid plates, scrape off a small amount of spores for colony PCR. Identification primers are 5680JDF and 5680JDR. The template for the positive control is the pKC1139-Δ5680 plasmid, and the template for the negative control is the A226 genome. The PCR product size is used as a reference. Figure 3 ), and obtained the ΔSACE_5680 deletion mutant.

[0042] Example 3

[0043] Construction of SACE_5680 gene complementation and overexpression strain:

[0044] The SACE_5680 gene was amplified using designed primers 5680-P1 and 5680-P2, and recovered by electrophoresis. The recovered SACE_5680 gene fragment was double-digested with pIB139 using NdeI and XbaI restriction enzymes, and then recovered. The SACE_5680 gene fragment was ligated to pIB139 using T4 DNA ligase, successfully obtaining the integrative plasmid pIB139-5680. Then, pIB139-5680 was introduced into ΔSACE_5680 protoplasts using PEG-mediated protoplast transformation. Preliminary screening was performed using apramycin, and PCR identification was conducted using apramycin resistance genes as the target. Figure 4 As shown, the obtained responsive strain was named ΔSACE_5680 / pIB139-5680.

[0045] Example 4

[0046] HPLC detection of fermentation products of *Rhodotorula sacchariformis*:

[0047] The erythromycin A content was determined by inoculating *Rhodotorula polyspora* on TSB medium and incubating at 30°C with shaking for 48 h. The culture was then transferred to R5 liquid medium and incubated at 30°C with shaking for 168 h. The fermentation broth was then extracted with an organic solvent, evaporated to dryness in a water bath, dissolved in 1 mL of methanol, and filtered through a 0.22 μm organic filter membrane. Finally, the erythromycin A content in the sample was determined by an instrument.

[0048] Example 5

[0049] Detection of mycelial biomass of Rhodotorula sacchariformis:

[0050] The ΔSACE_5680 mutant and A226 were inoculated into 30 mL of liquid TSB with the same inoculation amount. After culturing in a shaker at 30 °C for 48 h, they were transferred to R5 medium and cultured in a shaker at 220 rpm at 30 °C for 168 h. Samples were taken at different time points during the period, washed with anhydrous ethanol, dried and weighed. After the measurement, the bacterial biomass curve was plotted based on the experimental data.

[0051] Example 6

[0052] Transcriptional analysis of genes within the erythromycin biosynthesis gene cluster in ΔSACE_5680:

[0053] To investigate the transcriptional levels of genes related to the erythromycin synthesis gene cluster in strain ΔSACE_5680, an RT-qPCR experiment was performed (relevant primers are shown in Table 2). ΔSACE_5680 and A226 were fermented in R5 medium. After 24 h of fermentation, 1 mL of each fermentation broth was transferred to a homogenization tube and centrifuged at 12000 rpm for 15 min at 4 °C. After discarding the supernatant, 1 mL of Transzol reagent was added for homogenization and vortexing. Total RNA was then extracted using an RNA extraction kit and quantified using Nanodrop. RNA with an OD260 / 280 ratio between 1.9 and 2.2 was selected for subsequent experiments. Genomic DNA was digested from the RNA and promptly reverse transcribed into cDNA. Finally, using the reverse-transcribed cDNA as a template, an RT-qPCR kit was used to prepare the reaction system, and the transcriptional levels of genes related to the erythromycin synthesis gene cluster were detected using a qPCR instrument.

[0054] Example 7

[0055] Construction and HPLC detection of the high-yield erythromycin industrial strain WB / ΔSACE_5680:

[0056] The high-yielding erythromycin industrial strain WB was modified by deleting SACE_5680, and the correct strain was named WB / ΔSACE_5680. The fermentation products of the high-yielding WB strain and the deletion mutant WB / ΔSACE_5680 were then analyzed by HPLC. The mutant construction process and HPLC detection are as described in the above examples.

[0057] Example 8

[0058] Specific experimental results of the above embodiments:

[0059] 1. The deletion mutant ΔSACE_5680 showed increased erythromycin production compared to the original strain A226.

[0060] After 7 days of fermentation in the fermentation medium, the yield of erythromycin A was detected by HPLC. The results showed that the yield of ΔSACE_5680 was 22% higher than that of the starting strain A226 (see...). Figure 5 HPLC results showed that SACE_5680 is a negative regulator involved in erythromycin biosynthesis.

[0061] 2. SACE_5680 gene reversion and overexpression.

[0062] To verify that the increased erythromycin yield in the mutant ΔSACE_5680 was due to the deletion of the SACE_5680 gene, the SACE_5680 gene expression vectors pIB139-5680 and pIB139 (as controls) were introduced into the protoplasts of the ΔSACE_5680 mutant strain, obtaining revertant strains and empty vectors ΔSACE_5680 / pIB139-5680 and ΔSACE_5680 / pIB139. After PCR confirmation, the A226 and ΔSACE_5680 series mutant strains were subjected to shake-flask fermentation. HPLC results showed that the erythromycin A yield of ΔSACE_5680 was 22% higher than that of A226; the erythromycin A yield of the revertant strain ΔSACE_5680 / pIB139-5680 was basically restored compared to A226 (see...). Figure 6 HPLC results further indicate that the SACE_5680 gene can negatively regulate the biosynthesis of erythromycin A.

[0063] 3. Effects of SACE_5680 gene deletion on bacterial growth.

[0064] The cell dry weights of the ΔSACE_5680 mutant and the A226 strain were measured after 7 days of fermentation, and corresponding change curves were plotted. The results showed that the biomass of ΔSACE_5680 and A226 was not significantly different (see...). Figure 7 This suggests that the deletion of the SACE_5680 gene did not affect the primary metabolism of the bacteria.

[0065] 4. The transcription amount of genes within the erythromycin biosynthesis gene cluster increased in the ΔSACE_5680 mutant strain.

[0066] Compared with the originating strain A226, the transcription levels of ermE, eryAI, eryCI, eryK, eryBI, eryBIII, eryBIV, and eryBVI were significantly increased in the ΔSACE_5680 mutant. This indicates that SACE_5680 has a direct regulatory effect on the transcription of genes within the erythromycin biosynthesis gene cluster (see...). Figure 8 ).

[0067] 5. The modified industrial high-yield strain WB / ΔSACE_5680 increased the yield of erythromycin.

[0068] After constructing the WB / ΔSACE_5680 mutant strain, both the WB / ΔSACE_5680 mutant strain and the high-yield industrial strain WB were plated for activation. They were then inoculated separately into shake flasks containing industrial seed culture medium and cultured at 30℃ and 220 rpm for 2 days. Afterward, they were transferred to industrial fermentation medium and cultured for another 7 days. After fermentation, extraction and concentration were performed, and HPLC analysis showed that compared to the starting strain WB, the erythromycin yield of WB / ΔSACE_5680 was increased by 53% (see...). Figure 9 This indicates that the SACE_5680 gene in the high-yielding strain WB is also involved in regulating erythromycin production.

[0069] 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 *Rhodotorula sacchari* SACE_5680 The method for increasing erythromycin production through gene therapy is characterized by, Genetic engineering was used to induce the GntR family in *Rhodotorula polyspora* WB. SACE_5680 Gene deletion was performed to obtain a high-yield engineered strain of *Rhodotorula sacchariformis* for erythromycin production. The obtained high-yield engineered strain of *Rhodotorula sacchariformis* was then used for fermentation to produce erythromycin. The gene deletion was performed on the strain. SACE_5680 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The modified Rhodotorula sacchariformis according to claim 1 SACE_5680 The method for increasing erythromycin production through gene therapy is characterized by, The SACE_5680 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

2.

3. The modified Rhodotorula sacchariformis according to claim 1 SACE_5680 The method for increasing erythromycin production through gene therapy is characterized by, The SACE_5680 Gene products negatively regulate erythromycin biosynthesis.

4. A kind SACE_5680 The application of genetically modified saccharopolysporum erythromycetes is characterized by, The method constructed using any one of claims 1 to 3 SACE_5680 Genetically modified erythromycin polyspora is used in erythromycin production.

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