Method for improving erythromycin yield by modifying saccharopolyspora erythraea sace_7158 gene and application thereof

Abstract

This invention provides a method for modifying *Rhodotorula sacchari*. SACE_7158 A method to increase erythromycin production through gene editing involves knocking out *Rhodotorula sacchariformis* strain A226. SACE_7158 Genes were used to obtain a mutant strain of *Rhodotorula sacchari* that produces high levels of erythromycin, and the resulting strain was used for fermentation to produce erythromycin. SACE_7158 The gene sequence is shown in SEQ ID NO.1. This invention also provides... SACE_7158 Application of gene knockout-type *Rhodotorula glycopyrrolizum*. The advantages of this invention are: the discovery of a novel negative regulatory transcription factor, SACE_7158, involved in erythromycin biosynthesis, and the elimination of this gene knockout gene in the A226 genome of *Rhodotorula glycopyrrolizum*. SACE_7158 SACE_7158 A genetically engineered strain that produces high levels of erythromycin has been developed, providing technical support for increasing erythromycin yield in industrial production.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a method for modifying *Rhodotorula sacchari*. SACE_7158 Methods and applications of gene-based enhancement of erythromycin production. Background Technology

[0002] Actinomycetes are a group of Gram-positive bacteria that play an important role in both ecological function and biotechnology. They not only drive the organic matter cycle in nature but are also an important source of antibiotics and other bioactive natural products (such as pesticides, anticancer drugs, and immunosuppressants). Currently, more than two-thirds of antibiotics are produced by actinomycetes. However, the original antibiotic yield of naturally occurring actinomycete strains is generally low, requiring cumbersome screening and mutagenesis processes to obtain high-yielding strains that meet the requirements of industrial production. Traditional random mutagenesis breeding methods are not only time-consuming and labor-intensive with low screening efficiency but also cannot achieve precise and rational design of strains, becoming a key bottleneck restricting the improvement of efficiency in the industrial production of antibiotics.

[0003] Erythromycin is a macrolide antibiotic with broad-spectrum antibacterial effects, mainly produced by *Rhodotorula sacchariformis* (Saccharopolysporum). Saccharopolyspora erythraea Erythromycin, produced through fermentation, has erythromycin A as its main active ingredient. Derivatives such as clarithromycin, azithromycin, and roxithromycin have become commonly used preparations for clinical anti-infective treatment, and market demand continues to rise. Therefore, targeted modification of *Rhodotorula sacchariflora* using precise genetic engineering techniques to identify and regulate key genes involved in erythromycin biosynthesis, thereby increasing the erythromycin fermentation yield of industrial production strains, has significant economic and social value.

[0004] Transcriptional regulators are among the core factors regulating the metabolic pathways of *Rhodotorula sacchariflora* and determining its antibiotic fermentation yield. The genome of this strain contains multiple transcriptional regulator families, including TetR, Lrp, MarR, and LysR, with the TetR family of transcriptional regulators (TFRs) being the most numerous. Their regulatory functions cover multiple physiological and metabolic processes, including antibiotic biosynthesis, cell morphology differentiation, quorum sensing, and drug efflux. However, current research has only confirmed that a few TetR family factors, such as SACE_3986 and SACE_7301, participate in the regulation of erythromycin biosynthesis, and the relevant regulatory mechanisms differ among them. Although modifying these regulators and their targets has been proven to be an effective way to increase erythromycin yield, the functions of the vast majority of TetR family members in *Rhodotorula sacchariflora* have not yet been explored. Whether they participate in and how they regulate erythromycin biosynthesis remains unclear, failing to provide more effective references for the rational design of high-yielding erythromycin strains. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a modified Rhodotorula sacchariformis. SACE_7158 The method and application of gene-based enhancement of erythromycin yield aim to compensate for the lack of rationally designed target sites in existing high-yield erythromycin strains, and to discover novel negative regulatory genes involved in erythromycin biosynthesis, thereby achieving a significant increase in erythromycin yield.

[0006] The present invention solves the above-mentioned technical problems by adopting the following technical solutions: A modified Rhodotorula sacchariformis SACE_7158 A method to increase erythromycin production through gene therapy involves knocking out the TetR family gene in *Rhodotorula sacchariformis* strain A226 using genetic engineering. SACE_7158 Genes were used to obtain a mutant strain of *Rhodotorula sacchariformis* that produces high levels of erythromycin, and the resulting mutant strain was used for fermentation to produce erythromycin; SACE_7158 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0007] As one of the preferred embodiments of the present invention, the SACE_7158 The amino acid sequence encoded by the gene is shown in SEQ ID NO.2. As one of the preferred embodiments of the present invention, the SACE_7158 The gene expression product can directly and specifically bind to the promoter region of the erythromycin biosynthesis gene cluster, thereby inhibiting the expression of the erythromycin biosynthesis gene cluster. eryAI , ermE, eryBIII, eryBVI, eryCI, eryK Gene transcription negatively regulates the biosynthesis of erythromycin.

[0008] A sort of SACE_7158 The application of gene knockout glycopolysporid erythromycetes will utilize the strains obtained through the above methods. SACE_7158 Gene knockout saccharopolysporum erythromycin is used in erythromycin production.

[0009] The advantages of this invention compared to the prior art are: This invention uncovers a novel negative regulatory transcription factor, SACE_7158, involved in erythromycin biosynthesis, effectively addressing the lack of rationally designed targets in existing high-yield erythromycin strains. This transcription factor works by inhibiting... eryAI , ermE , eryBIII , eryBVI , eryCI , eryK Gene transcription negatively regulates erythromycin biosynthesis; this invention uses genetic engineering technology to knock out a gene in the genome of *Rhodotorula glycosporidis* A226. SACE_7158 Genes were successfully used to construct an engineered strain that produces high levels of erythromycin, providing technical support for increasing erythromycin production in industrial settings.

[0010] Specific experiments confirmed that knocking out *Rhodotorula sacchariformis* A226... SACE_7158The gene increased erythromycin A production by 28.4%; in Δ SACE_7158 Replacement in mutant strains SACE_7158 Gene, erythromycin production was restored; overexpression in A226 SACE_7158 The gene reduced erythromycin A production by 34.5%; this fully verifies that the transcription factor SACE_7158 can negatively regulate the biosynthesis of erythromycin. Attached Figure Description

[0011] Picture 1 It is a type of saccharopolysporum erythromycete. SACE_7158 Genomic location information map of the gene and its neighboring genes; Picture 2 It is Δ SACE_7158 The construction process of the mutant and the PCR identification results are shown in the figure (Figure A is Δ). SACE_7158 Schematic diagram of mutant construction; Figure B shows Δ SACE_7158 PCR identification of mutants, including those from the genome of *Rhodotorula sacchariformis*. SACE_7158 Partial sequence of the gene is replaced by the thiosporin resistance gene. tsr The replaced, including SACE_7158 A portion of the DNA fragment (600bp) was... tsr (1360bp) Replacement increases length to 1617bp; M: 5000bp DNA Marker). Picture 3 yes SACE_7158 PCR identification results of gene-complemented strain, complemented control strain, and overexpression strain (Figure A shows the Δ gene of the complemented strain). SACE_7158 PCR identification of pIB139-7158, 1: pIB139-7158, 2: Δ SACE_7158 ,3:Δ SACE_7158 / pIB139-7158; Figure B shows the replacement control strain Δ SACE_7158 PCR identification of pIB139, 1: pIB139, 2: Δ SACE_7158 ,3:Δ SACE_7158 Figure C shows the PCR identification of the overexpressing strain A226 / pIB139-7158, 1: pIB139-7158, 2: A226, 3: A226 / pIB139-7158; The amplified DNA fragment is the apramycin resistance gene. aac(3)IV ;M: 5000bp DNA Marker); Picture 4 yes SACE_7158 HPLC analysis of erythromycin A production in gene series strains (in the figure, "***" indicates...) P <0.001; "*" indicates P<0.05; "ns" indicates no significant difference); Picture 5 It is strain A226 and Δ SACE_7158 Phenotypic analysis of spore development, growth curves, and mycelial dry weight (Figure A shows the comparison of spore development; Figure B shows the measurement of growth curves; Figure C shows the measurement of mycelial dry weight). Picture 6 It is strain A226 and Δ SACE_7158 Transcriptional level analysis of related genes within the erythromycin synthesis gene cluster (in the figure, "***" indicates...) P <0.001; "**" indicates P <0.01; "*" indicates P <0.05; "ns" indicates no significant difference); Picture 7 This is an SDS-PAGE image of the SACE_7158 protein (in the image, 1: SACE_7158 protein eluted in 500mM imidazole buffer after pET28a-7158-induced expression; M: protein marker). Picture 8 It is a gene target site within the SACE_7158 protein and erythromycin synthesis gene cluster (promoter probe P). eryAI , ermE- eryCI-int P eryBVI P eryBIII and P eryK EMSA analysis chart. Detailed Implementation

[0012] 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.

[0013] 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.

[0014] Meanwhile, the *Escherichia coli* strains used in the following examples were cultured on liquid LB medium at 37ºC or on solid LB medium supplemented with 1.25% agar. *Rhodotorula glycosides*, a erythromycin-producing bacterium, was cultured on tryptone soybean broth (TSB), liquid R5 medium, or solid R5 medium supplemented with 2% agar at 30ºC.

[0015] In the following examples, PEG3350 and thiotetracycline were purchased from Sigma-Aldrich. Lysozyme, TES, and apramycin were purchased from Sangon Biotech. TSB, casein amino acids, yeast extract, and peptone were purchased from Oxoid. Glycine, agar, and other chemicals were purchased from a reagent company. Routine procedures for Escherichia coli and Rhodotorula sacchariformis were performed according to standard operating procedures. DNA synthesis and sequencing were outsourced to Sangon Biotech (Shanghai) Co., Ltd.

[0016] Table 1. The bacterial strains, plasmids, and their main properties involved in this invention.

[0017] Table 2 This invention relates to primers.

[0018] Example 1 SACE_7158 Gene-related information: Based on information from the KEGG database (https: / / www.kegg.jp / ), the genome of *Rhodotorula sacchariformis*... SACE 7158 See the location of its surrounding genes. Picture 1 .

[0019] SACE_7158 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2.

[0020] Example 2 SACE_7158 Construction of gene knockout mutants: SACE_7158 The gene knockout process is as follows Picture 2 As shown in A. Using primer pairs 7158-UF / R and 7158-DF / R in Table 2, and with *Rhodotorula sacchariflora* A226 genomic DNA as a template, the following results were amplified: SACE_7158 Homologous DNA fragments, 1.5 kb upstream and downstream of the gene, were then ligated into pUCTSRs. tsr Recombinant plasmid pUCTSRΔ was constructed on both sides of the gene. 7158 Using this plasmid as a template, primer pair 7158-F / R was used to amplify a plasmid containing... SACE_7158 Upstream and downstream homologous arms and tsrDNA fragments of the gene. Using a PEG-mediated method, the above fragments were introduced into protoplasts of *Rhodotorula sacchariflora* A226. Following linear fragment homologous recombination and selection for thiopeptide resistance, a gene was constructed. SACE_7158 Genes were tsr The recombinant engineered strain was replaced. Using primer pair 7158-VF / R, the above pUCTSRΔ was used. 7158 Using A226 genomic DNA as a negative control template, PCR identification was performed. The successfully identified engineered mutant strain was named Δ. SACE_7158 ( Picture 2 B).

[0021] Example 3 SACE_7158 Gene complementation and construction of overexpression strains: To further confirm SACE_7158 The regulatory role of genes in erythromycin production was investigated using primer pair 7158-CO-F / R from Table 2, with the A226 genome as a template. SACE_7158 Gene fragment. This fragment and the pIB139 plasmid were respectively... Nde I / Xba I double digestion, then T4 ligase was used to ligate SACE_7158 The gene was ligated into pIB139, and after screening for ampicillin resistance, PCR verification, and double enzyme digestion confirmation, the recombinant plasmid pIB139-7158 was successfully constructed. Then, under PEG3350-mediated transformation, plasmids pIB139-7158 and pIB139 were introduced into Δp0.05Cell, respectively. SACE_7158 The protoplasts were used to successfully construct the replenished strain Δ SACE_7158 / pIB139-7158 Picture 3 A) Compared with the replenishment control strain Δ SACE_7158 / pIB139 ( Picture 3 B). Simultaneously, plasmids pIB139-7158 and pIB139 were introduced into the protoplasts of A226, successfully constructing the overexpression strain A226 / pIB139-7158. Picture 3 C) and control strain A226 / pIB139.

[0022] Example 4 SACE_7158 HPLC detection of fermentation products from gene series strains: Take the starting strain A226 with good growth and SACE_7158 Gene series mutant strains (Δ SACE_7158, Δ SACE_7158 / pIB139、Δ SACE_7158Spores of A226 / pIB139-7158, A226 / pIB139, and A226 / pIB139-7158 were transferred to 50 mL of TSB medium and cultured at 30ºC with shaking for 48 h. The seed culture was then transferred to liquid R5 medium at a 10% inoculum and cultured for another 7 days under the same fermentation conditions. Erythromycin was obtained by chloroform extraction of the fermentation broth. The evaporated sample was dissolved in 1 mL of methanol, filtered, and the erythromycin A concentration of each sample was analyzed by HPLC.

[0023] Statistical results after HPLC detection are as follows Picture 4 As shown: Δ SACE_7158 The yield of erythromycin A was increased by 28.4% compared to the original strain A226. And in Δ SACE_7158 Replacement in mutant strains SACE_7158 The gene, whose erythromycin A production was restored, was also overexpressed in A226. SACE_7158 The gene reduced erythromycin A production by 34.5%. This indicates that SACE_7158 negatively regulates erythromycin production.

[0024] Example 5 A226、Δ SACE_7158 and Δ SACE_7158 Analysis of spore morphology, growth curve and mycelial dry weight of / pIB139-7158: Select the well-growing A226 and Δ SACE_7158 The spores were spread onto R5 solid plates, which were then incubated at 30ºC. Spore growth was observed and recorded every 24 hours. Results are as follows: Picture 5 As shown in A: Δ SACE_7158 The spore growth trend was basically the same as that of A226, with no obvious advance or delay.

[0025] Select the well-growing A226 and Δ SACE_7158 The spores were transferred to 50 mL of TSB medium and cultured with shaking at 30ºC. Samples of the culture medium were taken every 24 h, and the absorbance of the samples was measured at a wavelength of 600 nm. Based on the measured data, a graph was plotted between A226 and Δ... SACE_7158 The growth curve was obtained. Simultaneously, following the fermentation culture method of the strain in Example 4, samples of the fermentation broth were taken every 24 hours, the bacterial precipitate was collected by centrifugation, washed with anhydrous ethanol, dried, and the dry weight of the bacterial cells was measured. Based on the measured data, a graph was plotted between A226 and Δ. SACE_7158 The dry weight variation curve. The results are as follows: Picture 5 B and Picture 5 As shown in C: Compared to A226, Δ SACE 7158 The mycelial growth showed no significant change.

[0026] The results above indicate that, under the current experimental conditions, SACE_7158 does not affect the spore development and cell growth of *Rhodotorula sacchariformis*.

[0027] Example 6 A226 and Δ SACE_7158 Transcriptional level analysis of related genes: Following the fermentation culture method of the strain in Example 4, A226 and Δ were collected by centrifugation after 24 and 48 hours of fermentation. SACE 7158 The bacterial cell pellets were collected, and mRNA was extracted from the bacterial cells using the TransZol kit. After genomic DNA digestion and RNA reverse transcription, cDNA was obtained and analyzed by qRT-PCR using the primers listed in Table 2.

[0028] The results are as follows Picture 6 As shown: After 24 hours of cultivation, compared with A226, Δ SACE_7158 middle eryAI , ermE, eryBIII , eryBVI and eryCI The transcription levels increased by 1.6-fold, 1.7-fold, 1.5-fold, 1.5-fold, and 2.0-fold, respectively. Picture 6 A) After 48 hours of cultivation, Δ SACE_7158 middle eryAI , eryBVI , eryCI and eryK The transcription levels were increased by 1.5-fold, 1.6-fold, 1.9-fold, and 2.0-fold, respectively, compared to A226. Picture 6 B). This indicates that SACE_7158 can inhibit the erythromycin synthesis gene cluster. eryAI , ermE, eryBIII, eryBVI, eryCI, eryK Gene transcription negatively regulates the biosynthesis of erythromycin.

[0029] Example 7 EMSA experiment on SACE_7158 protein expression and its relationship with gene targets within the erythromycin synthesis gene cluster: Using the primer pair 7158-28a-F / R in Table 2, and with A226 genomic DNA as a template, the full-length amplified DNA was amplified by PCR. SACE_7158 Gene fragments, after Nde I / Hin After double digestion with dIII, the plasmid was ligated into the protein expression vector pET28a, and the recombinant plasmid pET28a-7158 was successfully constructed.

[0030] The above pET28a-7158 was transformed into Escherichia coli BL21(DE3) competent cells and cultured on LB solid plates containing kanamycin resistance. Single colonies were picked and expanded to obtain the SACE_7158 protein expression strain BL21 / pET28a-7158.

[0031] The above-mentioned BL21 / pET28a-7158 bacterial culture was transferred to kanamycin-resistant liquid LB medium and incubated at 37ºC for 12 h. Then, at a 1% inoculum size, it was transferred to 50 mL of the same resistance-containing liquid LB medium and cultured until the bacterial cell OD reached its maximum. 600 The concentration was approximately 0.6, and then IPTG was added to a final concentration of 0.5 mM. Expression was induced for 20 h at 16ºC and 180 rpm. The bacterial cells were collected by centrifugation and sonicated. The protein was purified using a nickel ion column, and the elution product in 500 mM imidazole buffer was analyzed by SDS-PAGE to obtain the SACE_7158 protein. Picture 7 ).

[0032] Using the primer pairs eryAI-F / R, ermE-CI-F / R, eryBIII-F / R, eryBVI-F / R, and eryK-F / R in Table 2, P was amplified and recovered using the A226 genome as a template. eryAI , ermE-eryCI-int P eryBVI P eryBIII and P eryK Promoter probe. The SACE_7158 protein was incubated with the above probe at 30ºC for 20 min, and then the activity of the reaction was analyzed by PAGE.

[0033] The results are as follows Picture 8 As shown: The SACE_7158 protein can specifically bind to P eryAI , ermE-eryCI-int P eryBVI P eryBIII and P eryK .

[0034] Based on comprehensive transcriptional analysis, SACE_7158 can directly and specifically bind to the promoter region of the erythromycin biosynthesis gene cluster, thereby inhibiting the erythromycin biosynthesis gene cluster. eryAI , ermE, eryBIII, eryBVI, eryCI, eryK Gene transcription negatively regulates the biosynthesis of erythromycin.

[0035] Based on this, the present invention has identified a novel negative regulatory transcription factor, SACE_7158, involved in erythromycin biosynthesis, effectively compensating for the lack of rationally designed target sites in existing high-yield erythromycin strains. Furthermore, by using genetic engineering technology to knock out the gene on the genome of *Rhodotorula glycosporidis* A226, SACE_7158 was successfully identified. SACE_7158 Genes can be used to successfully construct engineered strains that produce high levels of erythromycin, providing technical support for increasing erythromycin production in industrial settings.

[0036] 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_7158 The method for increasing erythromycin production through gene therapy is characterized by, The TetR family was knocked out in *Rhodotorula sacchariformis* strain A226 using genetic engineering methods. SACE_7158 Genes were used to obtain a mutant strain of *Rhodotorula sacchariformis* that produces high levels of erythromycin, and the resulting mutant strain was used for fermentation to produce erythromycin; SACE_7158 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The method according to claim 1, characterized in that, The SACE_7158 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

2.

3. The method according to claim 1, characterized in that, The SACE_7158 The gene expression product can directly and specifically bind to the promoter region of the erythromycin biosynthesis gene cluster, thereby inhibiting the expression of the erythromycin biosynthesis gene cluster. eryAI , ermE、eryBIII、eryBVI、eryCI、eryK Gene transcription negatively regulates the biosynthesis of erythromycin.

4. A kind SACE_7158 The application of gene knockout type saccharopolysporum erythromycetes is characterized by, The method constructed using any one of claims 1 to 3 SACE_7158 Gene knockout saccharopolysporum erythromycin is used in erythromycin production.

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