Tylosin a high-yield engineering strain and application thereof

Abstract

The application discloses a tylosin A high-yield engineering strain and application thereof, and belongs to the field of genetic engineering and fermentation technology. The strain is obtained by integrating a carbon mycose synthesis gene module (M14) overexpression cassette driven by a constitutive strong promoter into a genome and knocking out a negative regulation gene tylQ , so that the yield is greatly improved. Experiments show that the yield of the engineering strain in FFM culture medium reaches 1183 mg / L, which is 10 times that of a wild type; the yield can reach 1491.5 mg / L after adding alpha-ketoglutaric acid, and the genetic stability is good. The 5L fed-batch fermentation titer reaches 5.63 g / L. The application provides an efficient and stable industrial strain for large-scale production of tylosin A.

Description

Technical Field

[0001] This invention relates to engineered bacteria, specifically to a high-yield engineered strain of tylosin A and its applications. Background Technology

[0002] Tylosin is produced by Streptomyces freundii (… Streptomyces fradiae Tylosin, also known as *Streptomyces scintillans*, is an important 16-membered macrolide antibiotic. Due to its broad-spectrum antibacterial activity, it effectively inhibits Gram-positive bacteria, mycoplasma, and some Gram-negative bacteria, and is widely used in livestock and poultry farming and animal health. Structurally, tylosin uses a 16-membered tylosin core, with a disaccharide unit composed of mycaminose and mycarose linked at C-5, and a 6-deoxyallose monosaccharide unit linked at C-23. The structures of each component are shown in the following formula. Tylosin A, with its intact mycaminose, fully methylated allose, and C-19 aldehyde group, exhibits the strongest antibacterial activity and accounts for significantly higher proportions (over 80%) in the fermentation product than the other three components, making it a key indicator for evaluating tylosin potency. The biosynthesis and transfer efficiency of mycaminose, as the terminal glycosyl group of the disaccharide unit, is a key factor determining the yield of the highly active component, tylosin A.

[0003] .

[0004] The biosynthetic gene cluster (BGC) of tylosin is approximately 85 kb in size and contains at least 43 open reading frames, covering backbone synthesis, glycosylation modification, resistance determination, and a complex regulatory network. Within this regulatory system, tylP , tylQ , tylR , tylS and tylT Genes such as [names omitted] play a central role (Bate N, et al. Chem Biol, 1999, 6 (9): 617-624). Among them, tylQ The encoded TylQ protein is a typical transcriptional repressor that exerts a significant negative regulatory effect on the biosynthesis of tylosin by inhibiting the expression of the activator TylR.

[0005] Currently, traditional methods for increasing tylosin A production include physicochemical mutagenesis breeding and fermentation process optimization, but these methods suffer from problems such as high screening blindness and poor genetic stability of mutant strains. In recent years, researchers have attempted to modify strains through metabolic engineering techniques, such as overexpressing SAM synthase, methyltransferase, or positive regulatory genes. tylR and knocking out negative regulatory genes tylQHowever, most existing technologies are limited to the modification of single genes, with yield increases typically ranging from 10% to 30%. Furthermore, they often overlook the synergistic effect between precursor supply (such as glycosyl modules) and transcriptional regulation, making it difficult for modified strains to meet the potency requirements for industrial production.

[0006] Especially in Xinghai Bay Streptomyces ( Streptomyces xinghaiensis In this superior host, systematic enhancement of the complete carbamoyl synthesis module (M14), combined with the removal of transcriptional repression (knockout) tylQ The dual modification strategy of tylosin has not been reported. Therefore, developing a genetically engineered strain that can synergistically enhance the glycosylation pathway and block the negative regulatory pathway is of great scientific and economic value for significantly improving the fermentation level of tylosin and reducing industrial production costs. Summary of the Invention

[0007] Purpose of the invention: This invention addresses the shortcomings of existing technologies, such as the limited effect of single gene modification on increasing tylosin A production and the lack of a high-yield strain construction system for Streptomyces xinghai Bay. It provides a high-yield engineered strain of tylosin A constructed by synergistically strengthening the carbamolybdenum synthesis gene module and blocking the negative regulatory pathway, its construction method, and its application in fermentation production.

[0008] Technical solution: A high-yield engineered strain of tylosin A, characterized in that: an overexpression module of the carbamoylose synthesis gene module M14 is integrated into the genome of the strain, and the negative regulatory gene in the genome of the strain... tylQ Knocked out; the carbamoyl synthesis gene module M14 contains the following 9 key enzyme genes: tylAI , tylAII , tylO , tylCVI , tylCII , tylCIV , tylCIII , tylCV and tylCVII Their nucleotide sequences are shown in SEQ ID NO:1 to SEQ ID NO:9, respectively; Tylosin A.

[0009] The engineered strain is characterized in that: the source strain for each key enzyme gene in the carbamoylase synthesis gene module M14 is *Streptomyces xinghaiwanensis*. S. xinghaiensis ATCC 19609; The overexpression module contains a strong constitutive promoter kasOp*, which drives cascade transcription of multiple genes in the M14 module.

[0010] The engineered strain is characterized in that: the carboxylic acid synthesis gene module M14 consists of two fragments: fragment M14-1 contains the genes tylAI-tylAII-tylO-tylCVI, and fragment M14-2 contains the genes tylCII-tylCIV-tylCIII-tylCV-tylCVII.

[0011] The engineered strain is characterized in that: the negative regulatory gene tylQ The knockout site contains a streptomycin resistance gene. strA The strA The nucleotide sequence is shown in SEQ ID NO:12.

[0012] The engineered strain is characterized by being constructed according to the following steps: (1) Construct a recombinant co-expression plasmid of carbamolybdenum synthesis gene module M14, wherein the plasmid contains the kasOp* promoter and the nine key enzyme genes shown in SEQ ID NO:1 to SEQ ID NO:9; (2) The recombinant co-expression plasmid site was specifically integrated into the starting strain using the Escherichia coli-Streptomyces conjugation transfer method. S. xinghaiensis The genome of ATCC 19609 was used to obtain a mutant strain overexpressing the carbamoyl glycoside synthesis module. S. xinghaiensis M14-OE; (3) Constructing a system targeting negative regulatory genes tylQ The temperature-sensitive knockout plasmid was transformed into the strain obtained in step (2), and double crossover homologous recombination technology was used to block the knockout. tylQ Genes, to obtain the final engineered strain S. xinghaiensis M14-OE / ΔQ.

[0013] The application of the engineered strain in the fermentation production of tylosin A is characterized by the fact that the fermentation medium is selected from SM1, SM3, SM12, SM17, SM25, R5A, R3 or FFM.

[0014] The application is characterized in that the culture medium is FFM culture medium.

[0015] The application is characterized in that: the exogenous metabolic precursors in the FFM medium during fermentation are selected from aspartic acid, α-ketoglutarate, glutamic acid, malic acid, oxaloacetic acid, citric acid, succinic acid, pyruvic acid, alanine, lactic acid, glucose, fructose, vitamin B1, vitamin B2 or vitamin B6.

[0016] The application is characterized in that the exogenous metabolic precursor is α-ketoglutarate.

[0017] The application is characterized in that the concentration of the exogenous metabolic precursor α-ketoglutarate added during the fermentation process is 0.1~1.0 g / L.

[0018] The application is characterized in that the concentration of the exogenous metabolic precursor α-ketoglutarate is 0.5 g / L.

[0019] Specifically as follows: (1) Construct a recombinant co-expression plasmid of the carbamoylose synthesis gene module M14 and use the strong promoter kasOp* to drive the cascade transcription of multiple genes in the M14 module; (2) The co-expression plasmid sites were specifically integrated into the starting strain *Streptomyces xinghaiwan* using the *Escherichia coli*-streptomyces conjugation transfer method. S. xinghaiensis In the genome of ATCC 19609, a mutant strain overexpressing the carbamoylase synthesis module was obtained through screening with resistance markers. S. xinghaiensis M14-OE; (3) Constructing a system targeting negative regulatory genes tylQ Temperature-sensitive knockout plasmids; (4) Based on the above M14-OE strain, the negative regulatory gene was blocked using double crossover homologous recombination technology. tylQ Construct and obtain dual-modified engineered strains S. xinghaiensis M14-OE / ΔQ; (5) Using FFM medium for engineered bacteria S. xinghaiensis M14-OE / ΔQ was used for fermentation culture. During the fermentation process, 0.1-1.0 g / L (preferably 0.5 g / L) of the metabolic precursor α-ketoglutarate was added exogenously. Through fed-batch fermentation, the fermentation potency of tylosin A finally reached 5.63 g / L. (6) After fermentation, the fermentation broth was solvent extracted with four times the volume of ethyl acetate, and the target product tylosin A was obtained by separation and purification. Beneficial effects

[0020] (1) This invention is the first to be applied to Streptomyces cerevisiae in Xinghai Bay ( S. xinghaiensis This invention achieves co-overexpression of the carbamoyl glycoside synthesis gene module M14, directly eliminating the "metabolic bottleneck" in the biosynthesis and transfer of this glycoside at the overall metabolic pathway level, effectively overcoming the limitation of single-gene modification in existing technologies in terms of limited yield improvement. Experimental data show that the module overexpression mutant constructed in this invention... S. xinghaiensis The average yield of tylosin A in M14-OE reached 807.0 mg / L, which is about 7 times that of the wild-type starting strain, significantly enhancing the precursor supply capacity.

[0021] (2) Based on modular enhancement, this invention further removes negative regulatory genes. tylQ By synergistically integrating a dual strategy of "enhancing the positive synthetic pathway" and "relieving negative transcriptional repression," the resulting engineered strains... S. xinghaiensis The average yield of tylosin A in M14-OE / ΔQ was increased to 899 mg / L (approximately 7.8 times that of the wild-type strain); at the same time, due to the relief of the inhibition effect, the time to reach peak yield was shortened by about 24 hours compared to the starting strain, which greatly improved production efficiency.

[0022] (3) This invention, through the application of... S. xinghaiensis The M14-OE / ΔQ culture system was systematically optimized, establishing a suitable FFM fermentation medium that enabled tylosin A production to reach 1183 mg / L (approximately 10 times the optimal yield of the wild-type strain). Furthermore, by exogenously adding 0.5 g / L of α-ketoglutarate during fermentation, the advantages of the engineered strain in metabolic flux regulation were fully utilized, ultimately increasing the tylosin A potency to 1491.5 mg / L, a 13-fold increase compared to the wild-type strain. With the support of fed-batch fermentation, the tylosin A potency reached 5.63 g / L, demonstrating significant potential for industrial-scale production.

[0023] (4) The engineered strain constructed in this invention has excellent genetic stability. After 10 generations of continuous subculturing, the fluctuation range of its tylosin A production level is less than 5%, which meets the stringent requirements of industrial-scale fermentation for strain stability and reproducibility, and has significant economic value and application prospects. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the structural composition of the carbamoylose synthesis gene module M14. The diagram shows the genes contained in module M14. tylAI, tylAII, tylO, tylCVI, tylCII, tylCIV, tylCIII, tylCV, tylCVII ) and their order of arrangement; Figure 2 for Nde I and Xba The image shows the gel electrophoresis results of the recombinant plasmid pSET152-kasOp*-M14 verified by double enzyme digestion. The selected lane 1 in the image shows characteristic bands of approximately 9000 bp and 6000 bp, and the corresponding plasmids are the correctly identified recombinant plasmids. Figure 3 Recombinant strain S. xinghaiensis The figure shows the comparison of tylosin A production between M14-OE and wild-type strains. The figure shows the average tylosin A production of the two strains at different fermentation times (168 h, 192 h, 216 h) in SM12 medium. Error bars represent standard deviation (n=3). Figure 4 negative regulatory genes tylQ A diagram illustrating the knockout strategy, showing... tylQ The location of the gene in the genome, its upstream and downstream homologous arms, and the streptomycin resistance gene. strA The insertion site (nucleotide sequence as shown in SEQ ID NO: 12), and the changes in genome structure before and after knockout; Figure 5 To test the effects of different fermentation media on recombinant strains S. xinghaiensis The bar chart shows the effect of M14-OE / ΔQ on tylosin yield, comparing the tylosin A yield after fermentation for 264 h on eight different culture media: SM1, SM3, SM12, SM17, SM25, R5A, R3, and FFM. Figure 6 Recombinant strain S. xinghaiensis The genetic stability results of M14-OE / ΔQ after successive passages are shown in the figure. The figure shows the average yield of tylosin A in each generation of the strain during 10 consecutive passages. The error bars represent the standard deviation (n=3). Detailed Implementation

[0025] The technical solution of the present invention will be described in detail below through specific embodiments. These embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention in any way.

[0026] Example 1, Establishment of a method for detecting tylosin (standard curve method): Accurately weigh 30.0 mg of tylosin A standard, dissolve it in methanol, and dilute to 10 mL to prepare a stock solution with a concentration of 3.0 g / L. Serially dilute the stock solution with methanol to prepare a series of standard working solutions with concentrations of 1000, 500, 300, 250, 100, and 50 mg / L. Inject and analyze these solutions sequentially from lowest to highest concentration.

[0027] The HPLC analysis conditions are as follows: Chromatographic column: YMC-Pack ODS-A (150 mm × 4.6 mm, 5 μm); Mobile phase A: Ultrapure water (containing 0.1% formic acid); Mobile phase B: Acetonitrile (containing 0.1% formic acid); Detector parameters: Detection wavelength 286 nm, column temperature 30 ℃; Flow rate and injection volume: Flow rate 1.0 mL / min, injection volume 10 μL; Gradient elution program: 0-14 min: 90% A + 10% B; 14-15 min: 10% A + 90% B; A standard curve was plotted with tylosin A concentration (mg / L) on the x-axis and peak area on the y-axis, yielding the regression equation: y = 14463.91X - 84471.58, with a correlation coefficient R² = 0.9999, indicating that the method exhibits good linearity within the detection concentration range.

[0028] After fermentation, a quantitative amount of fermentation broth was taken, and four times its volume of ethyl acetate was added. The mixture was then shaken and extracted for 60 min. The upper ethyl acetate phase was collected, dried, and reconstituted with methanol. The ethyl acetate phase was then analyzed under the HPLC conditions described above. The actual yield of tylosin A in the fermentation broth was calculated using the regression equation described above.

[0029] Example 2, Construction of overexpression vector for carbamoyl glycoside synthesis-related gene modules: According to GenBank S. xinghaiensis Based on the genomic sequence information of ATCC 19609 (accession number: JNAD00000000.2), this invention designed and constructed a carbamoyl glycoside synthesis gene module M14 containing 9 genes. This module consists of two fragments: fragment M14-1 contains the gene... tylAI-tylAII-tylO-tylCVI (4347 bp), fragment M14-2 contains the gene tylCII-tylCIV-tylCIII-tylCV-tylCVII (5538 bp).

[0030] by S. xinghaiensis Using ATCC 19609 genomic DNA (purchased from the China Industrial Culture Collection Center CICC) as a template, primer pairs M14-1-F / R and M14-2-F / R (sequences shown in Table 1, with primers containing the desired fragments) were used. Nde I, Hind III and Xba The target fragment was obtained by PCR amplification of the restriction enzyme site (I).

[0031] Table 1 Primer Sequences .

[0032] The amplified fragment M14-1 was subjected to... Nde I / Hind III double digestion, fragment M14-2 was subjected to... Hind III / Xba After double digestion with enzyme I, it is combined with... Nde I / Xba I. The integrative vector pSET152 containing the kasOp* promoter was subjected to three-segment ligation. The ligation product was then transformed. E. coli DH5α competent cells were screened for resistance to 50 μg / mL apramycin sulfate and plasmids were extracted to obtain recombinant plasmids. Through... Nde I and XbaThe recombinant plasmid, confirmed by double enzyme digestion (characteristic bands of approximately 9000 bp and 6000 bp appearing) and identified correctly by sequencing, was named pSET152-kasOp*-M14.

[0033] Example 3: Construction and screening of mutant strains overexpressing carbamoyl glycoside synthesis module: The correctly constructed recombinant plasmid pSET152-kasOp*-M14 was transformed into donor-free bacteria. E. coli In ET 12567, donor bacterial resuspensions were prepared by screening on LB medium containing the corresponding antibiotic resistance markers. Simultaneously, [the following were collected / collected]. S. xinghaiensis Spores of ATCC 19609 were heat-shocked and used as recipient bacteria.

[0034] Donor bacterial suspension and recipient spore suspension were mixed in a specific ratio and spread onto WL-50 solid medium supplemented with 20 mM MgCl2, and co-cultured at 30 °C. After 17–18 hours of incubation, a sterile aqueous solution containing sodium nalidixate (to inhibit donor bacterial growth) and apramycin sulfate (to screen for resistant conjugates) was used for screening. Culture was continued until resistant monoclonal colonies appeared on the plates.

[0035] The resistance conjugates were picked and purified by streaking on MS medium containing apramycin. Genomic DNA of the purified strain was extracted and used as a template for PCR amplification of the apramycin resistance gene fragment on the vector using primer pair Apr-F / R (Apr-F: 5′-GTCATCAGCGGTGGA GTGC-3′; Apr-R: 5′-GCTCATGAGCTCAGCCAATCG-3′).

[0036] PCR amplification yielded a specific band of approximately 800 bp, demonstrating that the overexpression plasmid had been integrated into the target plasmid. S. xinghaiensis In the ATCC 19609 genome. The validating strain was named... S. xinghaiensis M14-OE.

[0037] Example 4, Evaluation of fermentation yield of strains overexpressing carbamoylose synthesis module: Recombinant strains S. xinghaiensis M14-OE and wild-type strains S. xinghaiensis After seed culture, ATCC19609 was transferred to SM12 fermentation medium at an inoculum of 2% and cultured with shaking at 30 °C and 230 rpm. Samples were taken periodically during fermentation, and the content of tylosin A was determined according to the method described in Example 1.

[0038] Detection data showed that the overexpression strain S. xinghaiensisThe M14-OE strain reached its peak yield at 288 h of fermentation, with an average tylosin A yield of 807.0 mg / L; while the wild-type strain reached its optimal yield at around 192 h, at only 114.0 mg / L. By recombinantly constructing and overexpressing the carbamoyl glycoside synthesis module M14, the yield of tylosin A was increased by 7.08 times compared to the wild-type strain, significantly improving the strain's biosynthetic capacity.

[0039] Example 5, Negative Regulatory Genes tylQ Knockout plasmid pKC1139- strA - ΔtylQ Construction: Constructing through homologous recombination strategy tylQ Gene knockout plasmids. S. xinghaiensis Using the ATCC 19609 genome as a template, PCR amplification was performed. tylQ The upstream homologous arm (nucleotide sequence as shown in SEQ ID NO:10) and downstream homologous arm (nucleotide sequence as shown in SEQ ID NO:11) of the gene; containing strA Using the gene plasmid as a template, streptomycin resistance gene marker fragments (nucleotide sequences as shown in SEQ ID NO:12) were amplified.

[0040] Overlap extension PCR was used, utilizing the homologous overlapping sequences between primers shown in Table 2, to extend the upstream homologous arm, strA The gene and its downstream homologous arms were spliced ​​together to obtain a knockout box fragment of approximately 5000 bp in length.

[0041] Table 2 Primer sequences used in overlap extension PCR technology .

[0042] The aforementioned knockout cassette fragment and the temperature-sensitive knockout vector pKC1139 were respectively subjected to... Eco RI and Hind After double digestion with enzyme III, ligation was performed overnight at 16 °C. The ligation product was then transformed. E. coli DH5α competent cells were used to extract plasmids, which were verified by enzyme digestion and sequencing to obtain the recombinant knockout plasmid, named pKC1139-. strA - ΔtylQ .

[0043] Example 6, tylQ Construction and screening of gene knockout strains: The recombinant plasmid pKC1139- strA - ΔtylQ After being transformed into donor bacteria, and with S. xinghaiensisM14-OE was used for conjugation transfer. Primary conjugates were obtained using a selection medium containing sodium nalidixinone and streptomycin. PCR verification confirmed that the plasmid had integrated into the genome via homologous recombination. The single-exchange strain was then cultured in antibiotic-free medium for relaxation, and plasmid loss and secondary recombination were induced at 37 °C using the temperature-sensitive properties of the vector. Streptomycin-sensitive strains were screened using a plate printing method, and... tylQ Gene flanking primers ΔtylQ -UP-F (5′-GATCACCTGTGTTCCACCGAG-3′) and ΔtylQ PCR verification was performed using the down-R (5′-GTGAGTGAGTCGCGAGTCGTC-3′) method. The results showed that the control strain amplified a fragment of approximately 1400 bp, while the recombinant strain amplified a fragment reduced to approximately 700 bp, demonstrating... tylQ The gene has been successfully knocked out. The strain that validated the correct overexpression and knockout of the negative regulatory gene will be named... S. xinghaiensis M14-OE / ΔQ.

[0044] Example 7, tylQ Evaluation of fermentation performance of knockout strains: Following the method in Example 4, the recombinant strain S. xinghaiensis M14-OE / ΔQ and control strains S. xinghaiensis Parallel fermentation experiments were conducted on M14-OE in SM12 medium. Periodic sampling and analysis during 144 h to 312 h revealed negative regulatory genes. tylQ The knockout further optimized the fermentation performance of the strain. The optimal fermentation period for M14-OE / ΔQ was 264 h, which was 24 h shorter than that for M14-OE. Its average yield of tylosin A reached 899 mg / L at 264 h, which was about 11% higher than the highest yield of M14-OE (807 mg / L), and about 7.8 times that of the wild-type original strain.

[0045] Example 8, Optimization of fermentation medium: Will S. xinghaiensisM14-OE / ΔQ seed culture was inoculated at a 2% inoculum into SM1, SM3, SM12, SM17, SM25, R5A, R3, and FFM liquid media, and fermented in parallel at 30 ℃ and 230 rpm for 264 h. Yield analysis showed that FFM medium exhibited the best compatibility, increasing tylosin A yield to 1183 mg / L, approximately 10 times the yield of the wild-type original strain. The FFM medium consisted of: 10.6 g / L fish meal, 16.5 g / L corn meal, 9.63 g / L gluten meal, 5.0 g / L cottonseed meal, 0.55 g / L betaine, 0.44 g / L (NH4)2HPO4, 0.0044 g / L CoCl2, 0.0033 g / L NiSO4, 2.2 g / L CaCO3, and 41 mL / L soybean oil. The pH was natural, and the medium was sterilized at 121 °C for 30 min.

[0046] Example 9, Effect of exogenous additives on tylosin A production: To further improve the production efficiency of the recombinant strain, 0.1 g / L of exogenous additives (selected from one of the following: aspartic acid, α-ketoglutarate, glutamic acid, malic acid, oxaloacetic acid, citric acid, succinic acid, pyruvic acid, alanine, lactic acid, glucose, fructose, vitamin B1, vitamin B2, and vitamin B6) were added to sterilized FFM medium via 0.22 μm filtration. S. xinghaiensis M14-OE / ΔQ seed culture was fermented at 30 ℃ and 230 rpm, and samples were taken periodically for testing during 312 h. The results showed that the potency of tylosin A after the addition of the above additives was aspartic acid (1106.2 mg / L), α-ketoglutarate (1288.9 mg / L), glutamic acid (956.2 mg / L), malic acid (981.6 mg / L), oxaloacetic acid (1218.0 mg / L), citric acid (1066.7 mg / L), succinic acid (875.4 mg / L), pyruvic acid (998.7 mg / L), alanine (1186.9 mg / L), lactic acid (1085.6 mg / L), glucose (1000.8 mg / L), fructose (1102.8 mg / L), vitamin B1 (1110.5 mg / L), vitamin B2 (1085.7 mg / L), and vitamin B6 (995.6 mg / L). Therefore, α-ketoglutarate had the greatest promoting effect on the fermentation potency of tylosin A.

[0047] Example 10, Investigation of the concentration of α-ketoglutaric acid: α-Ketoglutarate (α-KG) stock solution (250 mg / mL) was added to FFM medium to achieve final α-KG concentrations of 0 (control), 0.25 g / L, 0.5 g / L, and 1.0 g / L, respectively. Inoculation was then performed. S. xinghaiensis M14-OE / ΔQ seed culture was fermented at 30 ℃ and 230 rpm, with samples taken periodically over 312 h. The results showed that the addition of 0.5 g / L α-ketoglutarate had the most significant effect. At this concentration, the yield of tylosin A was consistently higher than that of the control group from 168 h onwards, reaching a peak of 1407.7 mg / L at 312 h, approximately 13 times the yield of the wild-type original strain.

[0048] Example 11, Investigation of shake-flask fermentation time: Add α-ketoglutarate to the FFM medium to a final concentration of 0.5 g / L. Inoculate. S. xinghaiensis M14-OE / ΔQ seed culture was fermented at 30 ℃ and 230 rpm, with samples taken periodically between 264 h and 360 h. The results showed that the fermentation potency of tylosin A was as follows: 1181.7 mg / L at 264 h, 1284.4 mg / L at 288 h, 1407.7 mg / L at 312 h, 1491.5 mg / L at 336 h, and 1490.8 mg / L at 360 h. Therefore, the optimal fermentation time was 336 h.

[0049] Example 12, Genetic stability test of the strain: To investigate the genetic stability of the recombinant strain, S. xinghaiensis M14-OE / ΔQ strains were inoculated into 2CM solid medium (soluble starch 10 g / L, tryptone 2 g / L, NaCl 1 g / L, (NH4)2SO4 2 g / L, K2HPO4 1 g / L, MgSO4 1 g / L, CaCO3 2 g / L, agar 12 g / L, pH 7.2) and passaged 10 times. Single colonies from each generation were randomly selected and inoculated into ISP2 liquid medium for amplification, followed by transfer to FFM liquid medium at a 2% inoculum. Fermentation was carried out at 30 ℃ and 230 rpm for 264 h, and the tylosin A yield was measured. The results showed that the tylosin A yield of the 1st to 10th generation strains remained stable between 1136.8 and 1185.9 mg / L, with fluctuations of less than 5%. This indicates that the recombinant strain... S. xinghaiensis M14-OE / ΔQ exhibits good genetic stability and has the potential for industrial fermentation.

[0050] Example 13, Fed-feed fermentation on a 5 L scale: To verify the recombinant strain S. xinghaiensis The production potential of M14-OE / ΔQ in industrial scale-up was demonstrated through fed-batch fermentation experiments using a 5L fully automated bioreactor.

[0051] The recombinant strain preserved on slant culture was inoculated into seed culture medium and cultured in shake flasks at 30 ℃ and 230 rpm to obtain seed liquid. Then, at an inoculation rate of 8%, the liquid was transferred to a 5L fermenter containing 3 L of FFM fermentation medium (containing 0.5 g / L α-ketoglutarate). Fermentation control parameters were set as follows: temperature 30 ℃, initial aeration rate 1.0 vvm, stirring speed 300 rpm. During fermentation, dissolved oxygen (DO) was maintained above 20% by linking stirring speed and aeration rate.

[0052] After 72 hours of fermentation, a fed-batch fermentation stage was initiated, with 240 mL of 2×FFM concentrated medium (containing 0.5 g / L α-ketoglutarate) added to the fermenter every 12 hours. Samples were taken periodically starting at 288 hours of fermentation. The fermentation broth was extracted with ethyl acetate and appropriately diluted according to the concentration. The tylosin A titer was then determined by HPLC. The results showed that the tylosin A yields at 288 h, 312 h, 336 h, and 360 h were 3.35 g / L, 4.51 g / L, 4.86 g / L, and 5.63 g / L, respectively. The experiment demonstrates that this engineered strain exhibits good tylosin A production capacity in a 5L scale fed-batch fermentation, with a final yield of 5.63 g / L, indicating promising prospects for industrial application.

Claims

1. A high-yield engineered strain of tylosin A, characterized by: The strain's genome contains an overexpression module of the carbamoylose synthesis gene module M14, and the strain's genome contains negative regulatory genes. tylQ Knocked out; the carbamoyl synthesis gene module M14 contains the following 9 key enzyme genes: tylAI , tylAII , tylO , tylCVI , tylCII , tylCIV , tylCIII , tylCV and tylCVII Their nucleotide sequences are shown in SEQ ID NO:1 to SEQ ID NO:9, respectively; Tylosin A.

2. The engineered strain according to claim 1, characterized in that: The source strain for each key enzyme gene in the carbamoyl synthesis gene module M14 is *Streptomyces xinghaiwanensis*. S. xinghaiensis ATCC 19609; The overexpression module contains a strong constitutive promoter kasOp*, which drives cascade transcription of multiple genes in the M14 module.

3. The engineered strain according to claim 1, characterized in that: The negative regulatory gene tylQ The knockout site contains a streptomycin resistance gene. strA The strA The nucleotide sequence is shown in SEQ ID NO:

12.

4. The engineered strain according to claim 1, characterized in that: The carbamoyl saccharide synthesis gene module M14 consists of two fragments: fragment M14-1 contains the genes tylAI-tylAII-tylO-tylCVI, and fragment M14-2 contains the genes tylCII-tylCIV-tylCIII-tylCV-tylCVII.

5. The engineered strain according to claim 1, characterized in that: It was constructed using the following steps: (1) Construct a recombinant co-expression plasmid of carbamolybdenum synthesis gene module M14, wherein the plasmid contains the kasOp* promoter and the nine key enzyme genes shown in SEQ ID NO:1 to SEQ ID NO:9; (2) The recombinant co-expression plasmid site was specifically integrated into the starting strain using the Escherichia coli-Streptomyces conjugation transfer method. S. xinghaiensis The genome of ATCC 19609 was used to obtain a mutant strain overexpressing the carbamoyl glycoside synthesis module. S. xinghaiensis M14-OE; (3) Constructing a system targeting negative regulatory genes tylQ The temperature-sensitive knockout plasmid was transformed into the strain obtained in step (2), and double crossover homologous recombination technology was used to block the knockout. tylQ Genes, to obtain the final engineered strain S. xinghaiensis M14-OE / ΔQ.

6. The application of the engineered strain according to any one of claims 1 to 5 in the fermentation production of tylosin A, characterized in that: The culture medium used for fermentation is selected from SM1, SM3, SM12, SM17, SM25, R5A, R3 or FFM.

7. The application according to claim 6, characterized in that, The culture medium is FFM medium.

8. The application according to claim 6, characterized in that: During fermentation, the exogenous metabolic precursors in the FFM medium are selected from aspartic acid, α-ketoglutarate, glutamic acid, malic acid, oxaloacetic acid, citric acid, succinic acid, pyruvic acid, alanine, lactic acid, glucose, fructose, vitamin B1, vitamin B2, or vitamin B6.

9. The application according to claim 8, characterized in that, The exogenous metabolic precursor is α-ketoglutarate.

10. The application according to claim 9, characterized in that, During fermentation, the concentration of the exogenous metabolic precursor α-ketoglutarate added is 0.1~1.0 g / L.

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