Streptomyces hygroscopicus ascomycetes subspecies and obtaining method thereof

Abstract

The invention discloses streptomyces hygroscopicus ascomycetes subspecies and an obtaining method thereof. Regulating genes fkbN, fkbR1 and fkbR2 on an ascomycin biosynthesis gene cluster of the streptomyces hygroscopicus ascomycetes subspecies ATCC 14891 and a gene fkbS participating in synthesis of an ascomycin precursor ethyl malonyl coenzyme A are subjected to biosynthesis to obtain the streptomyces hygroscopicus ascomycetes subspecies. And carrying out overexpression on a regulatory gene SH03686 of a leucine response regulatory protein family (Lrp) outside the gene cluster, and screening an ascomycin high-yield strain. According to the invention, after combined overexpression of genes fkbS and fkbN and overexpression of a gene fkbR2 are carried out by a strain SN9 and a strain R2-17 in starting bacteria, the transcriptional level of regulatory genes (fkbN, fkbR2) on an ascomycin biosynthesis gene cluster and related genes participating in synthesis of precursors ethyl malonyl coenzyme A (fkbS, fkbE), piperidine acid (fkbL) and DHCHC (fkbO) are improved, and finally, the ascomycin yield is improved.

Description

Technical Field

[0001] The invention relates to the technical field of Streptomyces hygroscopicus ascus subspecies, in particular to a Streptomyces hygroscopicus ascus subspecies and an acquisition method. Background Art

[0002] Ascomycin is a 23-membered macrolide antibiotic primarily produced by Streptomyces hygroscopicus subsp. ascomycin. Structurally similar to tacrolimus and rapamycin, it is a potent immunosuppressant widely used in the treatment of organ transplant rejection and some autoimmune diseases. Currently, it is primarily produced through microbial fermentation, but yields are low.

[0003] Currently, ascomycin is mainly produced through microbial fermentation. Although the biosynthetic method has many advantages, it still has disadvantages such as high production cost, low yield, long production cycle, complex intermediate products, and difficult separation. Among them, the main factor limiting the industrial large-scale production of ascomycin is the very low yield of wild strains. Now, some metabolic engineering strategies based on genetic modification have emerged, including enhancing the supply of specific precursors, eliminating or weakening by-product pathways, and gene overexpression. Although the yield of some has been improved, the stability of the strain is still poor.

[0004] To this end, we proposed a subspecies of Streptomyces hygroscopicus and a method for obtaining it. Summary of the Invention

[0005] The purpose of the present invention is to provide a Streptomyces hygroscopicus subspecies ascus and a method for obtaining it, so as to solve the problems raised by the above background technology.

[0006] To achieve the above objectives, the present invention provides the following technical solutions: a Streptomyces hygroscopicus subspecies ascus and a method for obtaining it, including strain SN9 and strain R2-17.

[0007] The second object of the present invention is to provide a method for obtaining Streptomyces hygroscopicus subsp. ascus, by overexpressing the regulatory genes fkbN, fkbR1, fkbR2 on the ascomycin biosynthesis gene cluster and the gene fkbS involved in the synthesis of the ascomycin precursor ethylmalonyl-CoA, as well as the regulatory gene SH03686 of the leucine response regulatory protein family (Lrp) outside the gene cluster in Streptomyces hygroscopicus subsp. ascus ATCC 14891, to screen for ascomycin high-yielding strains.

[0008] Further, specifically:

[0009] S1. First, a series of overexpression plasmids are constructed, and then these plasmids are transferred into Streptomyces hygroscopicus subsp. ascus by conjugation to obtain the corresponding ascomycin genetically engineered bacteria;

[0010] S2. The constructed ascomycin genetically engineered bacteria were then fermented in shake flasks to obtain the strains SN9 and R2-17 with the highest yields;

[0011] The fermentation processes of S3, strain SN9, strain R2 and the starting strain were compared.

[0012] Further, the series of overexpression plasmids include gene fkbS overexpression plasmid pIB139-fkbS, gene fkbS and fkbN combined overexpression plasmid pIB139-fkbSN, gene fkbS and fkbR1 combined overexpression plasmid pIB139-fkbSR1, gene fkbR2 overexpression plasmid pIB139-fkbR2, ​​and gene SH03686 overexpression plasmid pIB139-SH03686.

[0013] The third object of the present invention is to provide a Streptomyces hygroscopicus subspecies ascus for use in promoting the improvement of ascomycin production.

[0014] Further, after strains SN9 and strain R2-17 jointly overexpressed the genes fkbS and fkbN and overexpressed the gene fkbR2 in the starting bacteria, the transcription levels of the regulatory genes (fkbN, fkbR2) on the ascomycin biosynthesis gene cluster and the related genes involved in the synthesis of precursors ethylmalonyl-CoA (fkbS, fkbE), pipecolic acid (fkbL), and DHCHC (fkbO) were increased, ultimately promoting the increase in ascomycin production.

[0015] Compared with the prior art, the beneficial effect of the present invention is that after strain SN9 and strain R2-17 jointly overexpress the genes fkbS and fkbN and overexpress the gene fkbR2 in the starting bacteria, the transcription levels of the regulatory genes (fkbN, fkbR2) on the ascomycin biosynthesis gene cluster and the related genes involved in the synthesis of precursors ethylmalonyl-CoA (fkbS, fkbE), pipecolic acid (fkbL), and DHCHC (fkbO) are increased, ultimately promoting the increase in ascomycin production. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the result of the first natural selection and fermentation of strain SN9;

[0017] Figure 2 This is the result of the second natural selection fermentation of strain SN9;

[0018] Figure 3 This is the result of repeated fermentation of strain SN9-11-2;

[0019] Figure 4 This is the result of the third natural selection fermentation of strain SN9;

[0020] Figure 5 This is the result of the first natural selection and fermentation of strain R2-17;

[0021] Figure 6 This is the result of repeated fermentation of strain R2-17-3;

[0022] Figure 7 This is the result of the second natural selection fermentation of strain R2-17;

[0023] Figure 8 This is a comparative trend chart of fermentation process parameters between the high-yield strain SN9-11-2-7 and the starting strain WT;

[0024] Figure 9 Schematic diagram comparing gene transcription levels of high-yield strain SN9-11-2-7 and starting strain WT;

[0025] Figure 10 This is a schematic diagram of the comparative trend of fermentation process parameters between the high-yield strain R2-17-3-10 and the starting strain WT;

[0026] Figure 11 Schematic diagram comparing gene transcription levels of high-yield strain R2-17-3-10 and starting strain WT. DETAILED DESCRIPTION

[0027] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0028] The present invention provides a technical solution: a subspecies ascocarpus hygroscopicus and a method for obtaining it.

[0029] The authors screened high-ascomycin-producing strains by overexpressing the regulatory genes fkbN, fkbR1, and fkbR2 within the ascomycin biosynthesis gene cluster, fkbS involved in the synthesis of the ascomycin precursor ethylmalonyl-CoA, and SH03686, a leucine-responsive regulatory protein (Lrp) family regulatory gene, in Streptomyces hygroscopicus subsp. ascus ATCC 14891. The factors contributing to the increased ascomycin production were analyzed by measuring fermentation parameters and transcript levels of related genes in the high-yielding strains compared to the starting strain.

[0030] First, a series of overexpression plasmids were constructed, including the fkbS overexpression plasmid pIB139-fkbS, the fkbS and fkbN combined overexpression plasmid pIB139-fkbSN, the fkbS and fkbR1 combined overexpression plasmid pIB139-fkbSR1, the fkbR2 overexpression plasmid pIB139-fkbR2, ​​and the SH03686 overexpression plasmid pIB139-SH03686. These plasmids were then transferred into Streptomyces hygroscopicus subsp. ascus by conjugation to obtain the corresponding ascomycin genetically engineered strains.

[0031] The constructed ascomycin genetically engineered bacteria were then fermented in shake flasks, and the two strains with the highest yields, SN9 and R2-17, were subjected to multiple natural selections. While ensuring high yields, the stability of the high-yield strains was further improved. After three consecutive natural selections, the yield of strain SN9 increased from 120% to 234%. After two consecutive natural selections, the final yield of strain R2-17 increased by 34% compared to the starting strain. Finally, the two excellent strains SN9-11-2-7 and R2-17-3-10 with increased ascomycin production and good stability were screened out, which were significantly increased by 134% and 34% compared to the starting strains, respectively, with yields reaching 1226.38 mg / L and 686.47 mg / L, respectively.

[0032] Finally, the fermentation processes of the high-yielding strains (SN9-11-2-7 and R2-17-3-10) and the starting strain were compared, revealing differences in FK520 synthesis rate, pH, bacterial concentration, glycerol consumption rate, amino nitrogen level, and ammonium ion concentration. RT-qPCR was used to measure the transcription levels of genes in the ascomycin biosynthesis gene cluster at 48, 72, and 96 hours. It was found that the transcription levels of genes fkbS, fkbN, and fkbL in the high-yielding strain SN9-11-2-7 increased by 0.57-fold, 2.15-fold, and 9.97-fold, respectively, at 72 hours of fermentation, and genes fkbE, fkbO, and fkbR2 increased by 6.26-fold, 2.84-fold, and 1.13-fold, respectively, at 48 hours. In the high-yield strain R2-17-3-10, the expression of genes fkbN and fkbL increased by 13.24-fold and 4.48-fold, respectively, after 48 hours of fermentation. At 72 hours, the expression of genes fkbR2, ​​fkbO, and fkbE increased by 4.92-fold, 5.62-fold, and 4.16-fold, respectively. This suggests that the combined overexpression of genes fkbS and fkbN, as well as overexpression of gene fkbR2 in the starting strain, increased the transcription levels of regulatory genes (fkbN, fkbR2) within the ascomycin biosynthesis gene cluster and genes involved in the synthesis of the precursors ethylmalonyl-CoA (fkbS, fkbE), pipecolic acid (fkbL), and DHCHC (fkbO), ultimately promoting increased ascomycin production.

[0033] A series of constructed overexpression and knockout plasmids (pIB139-fkbS, pIB139-fkbSN, pIB139-fkbSR1, pIB139-fkbR2, ​​pIB139-SH03686, pKC1139-DfkbR2, ​​pC2-sgRNA1-5-HA, and pC2-sgRNA1-7-HA) were transformed into the starting strain WT using the intergeneric conjugative transfer method between Streptomyces hygroscopicus and Escherichia coli ET12567. The resulting conjugates were then verified by colony PCR and sequencing according to the conjugative verification procedures. After conjugative transfer of all overexpression plasmids, numerous conjugates grew on the conjugative transfer plates.

[0034] Screening of Ascomycin Genetically Engineered Strains

[0035] More than a dozen strains of the five different genetically engineered bacteria were randomly selected for shake flask fermentation. The fermentation results were always high and low, indicating that the yield of the genetically engineered strains may not always increase due to strain impurity and different positions of gene integration in the genome. Therefore, it is necessary to screen again through natural selection to obtain excellent strains with increased and stable yields.

[0036] The primary purpose of natural selection in this study is to ensure the high productivity of the high-yield strain and improve its stability on the basis of obtaining a high-yield strain. Therefore, in order to measure the effect of natural selection, the present invention defines two indicators: high-yield index and stability index. The high-yield index refers to the proportion of strains with a yield higher than WT after natural selection among all strains; the stability index refers to the proportion of strains with an average yield approximately equal to the average yield among all strains after natural selection. When the average yield is less than 200%, the yield in the interval of (average yield -10%, average yield +10%) is considered to be approximately equal to the average yield. When the average yield is greater than 200%, the yield in the interval of (average yield -20%, average yield +20%) is considered to be approximately equal to the average yield. The specific calculation method is shown in formula (4-1), formula (4-2) and formula (4-3). When both the high-yield index and the stability index reach 70%, it can be considered that the high productivity and stability of the strain meet the requirements, and no further selection is required, that is, a high-yield and stable strain is successfully bred, and subsequent experiments can be carried out on the strain with the highest yield.

[0037] Because SN9 showed the largest improvement among all the genetically engineered bacteria, followed by R2-17, which were 63% and 28% higher than the control strain WT, respectively, only SN9 and R2-17 were subjected to multiple natural selections until a high-yield and relatively stable strain was selected.

[0038]

[0039] Continuous natural selection of SN9 strain

[0040] The seed liquid of strain SN9 was diluted and spread according to the natural selection and preservation of the above strains. Eleven single colonies with good appearance, large size and good spore production were selected from the solid plate and named SN9-1 to SN9-11 respectively. They were then streaked on solid culture medium for expansion culture. After the spores on the plate matured and turned gray, the selected strains SN9-1 to SN9-11, the strain SN9 before selection and the starting strain WT were fermented according to the shake flask fermentation culture. After 5 days of fermentation, the yield of FK520 was detected by HPLC, and the results are as follows Figure 1 shown.

[0041] from Figure 1 It can be seen that after natural selection, the yield of 5 of the 11 strains was higher than that of the starting strain WT, with a high-yield index of 45%. Among them, SN9-11 with the highest yield increased by 20%, which was significant. The average yield of the 11 strains after natural selection was 99%, of which 7 strains had a yield approximately equal to the average yield (i.e., the yield was in the range of 89-109%), and the stability index was 64%. The stability index increased by 4% compared to before natural selection (60%), but the high-yield index and stability index have not yet reached 70%, that is, a strain that is both stable and high-yielding has not yet been screened. Therefore, the strain SN9-11 with the highest yield in this fermentation was subjected to natural selection again.

[0042] The strain SN9-11 was diluted and spread to separate, and the strains SN9-11-1 to SN9-11-10 after selection, the strain SN9-11 before selection, and the starting strain WT were fermented. The results are as follows Figure 2 As shown. Figure 2 It can be seen that: the yield of 3 out of the 10 strains after natural selection was higher than that of the starting strain WT, and the high-yield index was 30%. Among them, SN9-11-2 with the highest yield increased by 93%, which was significant. The average yield of the 10 strains after natural selection was 105%, of which 6 strains had a yield approximately equal to the average yield (that is, the yield was within the range of 95-115%), and the stability index was 60%. Since the high-yield index and the stability index still did not reach 70%, that is, a strain that was both high-yielding and stable had not yet been screened. And because the strain SN9-11-2 was particularly significantly improved compared to the starting strain WT, it was first subjected to repeated fermentation verification (the results are as follows Figure 3 As shown) and then natural selection. Figure 3 It can be seen that the results of repeated verification show that the yield of SN9-11-2 is 42% higher than that of the starting strain WT. Because each batch of fermentation is affected by many factors, resulting in different fermentation results, it can be shown that SN9-11-2 is indeed a high-yield strain.

[0043] The strain SN9-11-2 was diluted and spread to separate, and the strains SN9-11-2-1 to SN9-11-2-10 after selection, the strain SN9-11-2 before selection, and the starting strain WT were fermented. The results are as follows Figure 4 shown.

[0044] from Figure 4 It can be seen that the yield of strain SN9-11-2 is much higher than that of the starting strain WT, and the parallelism is also good, which once again shows that SN9-11-2 is indeed a high-yield strain. The yields of the 10 strains after natural selection are all higher than those of the starting strain WT, with a high-yield index of 100%. Among them, SN9-11-2-7 with the highest yield increased by 134%, and SN9-11-2-8 with the lowest yield also increased by 53%. The average yield of the 10 strains after natural selection was 206%, of which 7 strains had a yield approximately equal to the average yield (ie, the yield was in the range of 186-226%), and the stability index was 70%. Both the high-yield index and the stability index reached 70%, indicating that an excellent strain that is both high-yielding and stable has been successfully bred. Therefore, there is no need to continue breeding, and the fermentation process of the strain with the highest yield, SN9-11-2-7, and the starting strain WT will be compared.

[0045] The results of three natural selections of the above strain SN9 are summarized in Table 1 below.

[0046] Table 1 Three natural breeding results of strain SN9

[0047]

[0048] Continuous natural selection of strain R2-17

[0049] The seed solution of strain R2-17 was diluted and spread for separation, and 10 single colonies were selected and named R2-17-1 to R2-17-10. The selected strains R2-17-1 to R2-17-10, the strain R2-17 before selection, and the starting strain WT were fermented. The results are as follows Figure 5 shown.

[0050] from Figure 5It can be seen that: the yield of 5 out of the 10 strains after natural selection was higher than that of the starting strain WT, and the high-yield index was 50%. Among them, R2-17-3 with the highest yield increased by 64%, which was significant. The average yield of the 10 strains after natural selection was 104%, and the yield of 5 of them was approximately equal to the average yield (that is, the yield was in the range of 94-114%), and the stability index was 50%. Since both the high-yield index and the stability index did not reach 70%, that is, a strain that was both high-yielding and stable had not yet been screened. And because the strain R2-17-3 was particularly significantly improved compared to the starting strain WT, it was continued to be repeatedly fermented for verification (the results are as follows Figure 6 Then natural selection occurs.

[0051] from Figure 6 It can be seen that the results of repeated verification showed that the yield of R2-17-3 was 52% higher than that of the starting strain WT. Because each batch of fermentation is affected by many factors, resulting in different fermentation results, it shows that R2-17-3 is indeed a high-yield strain.

[0052] The seed liquid of strain R2-17-3 was diluted and spread according to the natural selection and preservation of the strain above. Ten single colonies with good appearance, large size and good spore production were selected from the solid plate and named R2-17-3-1 to R2-17-3-10 respectively. They were then streaked onto solid culture medium for expansion culture. After the spores on the plate matured and turned gray, the selected strains R2-17-3-1 to R2-17-3-10, the unselected strain R2-17-3, and the starting strain WT were fermented according to the shake flask fermentation culture in 2.2.2 above. After 5 days of fermentation, the yield of FK520 was detected by HPLC. The results are as follows Figure 7 shown.

[0053] from Figure 7 It can be seen that the yields of the 10 strains after natural selection were all higher than those of the starting strain WT, with a high-yield index of 100%. Among them, R2-17-3-10, which had the highest yield, increased by 34%. The average yield of the 10 strains after natural selection was 119%, and the yields of 8 of them were approximately equal to the average yield (i.e., the yields were within the range of 109-129%), with a stability index of 80%. Both high productivity and stability reached 70%, indicating that a high-yield and stable strain was successfully bred. Therefore, no further breeding was required, and the fermentation process of the strain with the highest yield, R2-17-3-10, and the starting strain WT were subsequently compared.

[0054] The results of the two natural selections of the above strain R2-17 are summarized in Table 2 below.

[0055] Table 4.2 Two natural breeding results of strain R2-17

[0056]

[0057] Comparison between high-yield strain SN9-11-2-7 and starting strain WT

[0058] Comparison of fermentation process parameters between SN9-11-2-7 and WT

[0059] The high-yield strain SN9-11-2-7 and the starting strain WT were fermented in shake flasks simultaneously. The FK520 yield, pH, bacterial concentration, glycerol, amino nitrogen, and ammonium ion of the two strains were measured at 48h, 72h, 96h, and 120h. The fermentation process parameters of the two strains were compared as follows: Figure 8 shown.

[0060] The changes in FK520 production over time during the fermentation process show that both the starting strain WT and the high-yielding strain SN9-11-2-7 began to synthesize FK520 at 48 hours, and at this time, SN9-11-2-7 produced more FK520 than WT. The high-yielding strain SN9-11-2-7 was in a rapid production phase from 48 to 120 hours, and the production rate continued to increase, from 3.3 mg / L / h to 7.1 mg / L / h and then to 12.9 mg / L / h, with the final yield reaching 977 mg / L, a significant increase of 77% over the starting strain WT. Therefore, from the perspective of the FK520 production trend, the difference between the two is that the production rate of SN9-11-2-7 is slower than that of WT in the early fermentation period (48-72 hours), while the production rate of SN9-11-2-7 continues to increase in the middle fermentation period (72-96 hours) and the late fermentation period (96-120 hours) and is far higher than that of WT, showing a clear difference.

[0061] The changes in pH over time during the fermentation process show that during the 48-96h period, the pH of the two strains was not much different, basically within the range of 4.5-5.0, which should be the pH range of the strains during the toxin production period. During the 96-120h period, there were obvious differences between the two strains: WT suddenly rose rapidly to around 6.2, which corresponds to the low FK520 production rate of WT in the later stage; at this time, the pH of the high-yielding strain SN9-11-2-7 was still maintained at around 5.0, indicating that even in the late fermentation stage, it was still in the toxin production stage, and there was still a large amount of organic acid in the cell that could be used for FK520 synthesis, which corresponds to the still high production rate of SN9-11-2-7 in the late fermentation stage.

[0062] The changes in bacterial concentration over time during the fermentation process show that the trends of the two strains are similar, both showing a trend of first increasing and then decreasing. The bacterial concentration reaches its maximum at 72 hours, and at this time the bacterial concentration is around 50%. Then it drops rapidly from 72 to 96 hours, and the rate of decline slows down from 96 to 120 hours. The only difference is that SN9-11-2-7 decreases faster than WT during the decline phase. Ultimately, the bacterial concentration of SN9-11-2-7 drops to around 30%, while the bacterial concentration of WT drops to around 37%. Therefore, from the perspective of bacterial concentration change trends, the difference between the two strains is not much, both reaching a maximum bacterial concentration of around 50% at 72 hours, except that the bacterial concentration of the high-yield strain SN9-11-2-7 is slightly lower than that of WT at the end of the final fermentation.

[0063] The temporal changes in glycerol levels during fermentation revealed that SN9-11-2-7 had higher glycerol levels at 48 hours, indicating that it consumed less glycerol during the early growth phase. Between 48 and 120 hours, SN9-11-2-7 consumed glycerol more slowly than WT, with the WT maintaining a roughly constant glycerol consumption rate of 0.065% / h and the SN9-11-2-7 maintaining a roughly constant glycerol consumption rate of 0.055% / h. During the glycerol production phase, both strains consumed glycerol at a roughly constant rate, with the high-yielding strain, SN9-11-2-7, utilizing glycerol more slowly than the WT.

[0064] The temporal changes in amino nitrogen during the fermentation process show that at 48 hours, SN9-11-2-7 had a higher amino nitrogen level, indicating that it consumed less amino nitrogen in the early growth phase. Combined with the fact that glycerol consumption was also lower than that of the WT, the bacterial concentration from 48 to 72 hours was essentially the same as that of the WT. This may indicate that the high-yielding strain has a lower demand for nutrients such as carbon and nitrogen sources, making it easier to grow than the starting strain WT. From 48 to 120 hours, both strains showed an upward trend and their growth rates continued to increase, indicating that the production rate of amino nitrogen in both strains was higher than the consumption rate. The amino nitrogen level of SN9-11-2-7 was consistently higher than that of the WT. Therefore, judging from the trend of amino nitrogen changes, the amino nitrogen level of the high-yielding strain SN9-11-2-7 during the production phase was consistently higher than that of the WT, which may be more conducive to the synthesis of FK520 and an important reason for the higher yield of SN9-11-2-7.

[0065] The temporal changes in ammonium ion concentrations during fermentation revealed that at 48 h, the ammonium ion concentrations of SN9-11-2-7 and WT were 0.346 mmol / L and 0.335 mmol / L, respectively, both less than 1 mmol / L. This indicates that both strains had essentially consumed all the ammonium sulfate in the culture medium by 48 h, a phenomenon that persisted until 96 h. From 96 to 120 h, the ammonium ion levels of the two strains differed significantly: while both strains increased, the increase in the starting strain WT was greater, reaching a final concentration of 6.215 mmol / L. Considering that glycerol was almost completely depleted at this time, this may be due to insufficient carbon source in the late stage of WT, which used amino acids as a carbon source, releasing ammonium ions, leading to an increase in ammonium ion concentration, which corresponded to the sudden and substantial increase in pH. In contrast, the high-yielding strain SN9-11-2-7 showed only a slight increase, with a final concentration of only 0.706 mmol / L, indicating that lower ammonium ion levels are more conducive to the production of FK520. Therefore, judging from the trend of ammonium ion changes, the ammonium ion level of the high-yield strain SN9-11-2-7 during the toxin production stage was always lower than that of the WT, which may be more conducive to the synthesis of FK520 and is an important reason for the higher yield of SN9-11-2-7.

[0066] Comparison of gene expression levels between SN9-11-2-7 and WT

[0067] Total RNA was extracted from the bacterial pellets of the high-yield strain SN9-11-2-7 and the starting strain WT at 48h, 72h, and 96h, and then the transcription levels of genes such as fkbN, fkbR2, ​​fkbS, fkbE, fkbO, and fkbL in the FK520 biosynthetic gene cluster were measured by RT-qPCR to analyze the differences between the two at the gene expression level. The results are as follows Figure 9 shown.

[0068] From the transcription levels of the gene fkbN at different time points, it can be seen that both WT and SN9-11-2-7 showed a trend of first increasing and then decreasing, and the expression levels were the highest at 72h, which were 3.30 times and 5.45 times, respectively. At 48 and 72h, the expression level of the gene fkbN in SN9-11-2-7 was significantly higher than that of WT, increasing by 0.72 times and 2.15 times, respectively. Because fkbN was co-overexpressed in strain SN9-11-2-7, it shows that the gene doubling strategy can achieve the increase in the expression level of this gene, and the gene fkbN positively regulates the biosynthesis of FK520, so its increased expression level at 48 and 72h may be an important reason for the increase in SN9-11-2-7 yield.

[0069] The transcription levels of the fkbS gene at different time points showed that after combined overexpression of fkbS and fkbN, fkbS transcript levels increased significantly by 0.57-fold at 72 hours. Like fkbN, the maximum increase was reached at 72 hours. fkbS encodes crotonyl-CoA reductase, which catalyzes the conversion of crotonyl-CoA to ethylmalonyl-CoA. Therefore, increased fkbS expression helps increase the supply of the precursor ethylmalonyl-CoA, thereby improving FK520 production.

[0070] The transcriptional levels of the fkbR2 gene at different time points showed that the WT showed an upward and then downward trend, reaching its highest expression level at 72 hours, 1.39 times that of 48 hours. In contrast, the expression level of the SN9-11-2-7 gene reached its maximum at 48 hours and then showed a downward trend. At 48 hours, the expression level of the fkbR2 gene in SN9-11-2-7 was 1.13 times higher than that in the WT.

[0071] Transcription levels of the fkbE gene at different time points revealed a significant 6.26-fold increase in SN9-11-2-7 compared to WT at 48 hours. fkbE encodes a putative homolog of L-carnitine dehydratase, catalyzing the conversion of trihydroxybutyryl-CoA to crotonyl-CoA, ultimately forming ethylmalonyl-CoA. It is a target gene of the regulatory gene fkbN. Increased fkbE expression also promotes the formation of the precursor ethylmalonyl, ultimately promoting the synthesis of FK520.

[0072] Comparison between high-yield strain R2-17-3-10 and starting strain WT

[0073] Comparison of fermentation process parameters between R2-17-3-10 and WT

[0074] The fermentation process parameters of the high-yield strain R2-17-3-10 and the starting strain WT are compared as follows Figure 10 shown.

[0075] The temporal changes in FK520 production during fermentation revealed that both the starting strain WT and the high-yielding strain R2-17-3-10 began producing FK520 at 48 hours, with R2-17-3-10 producing more than the WT at this point. The production rate of strain R2-17-3-10, however, initially decreased and then increased, from 5.2 mg / L / h to 4.1 mg / L / h and then to 7.1 mg / L / h, ultimately reaching 780 mg / L, a significant 41% increase over the starting strain WT. Therefore, the difference between the two strains in terms of FK520 production trends is that R2-17-3-10's production rate was slower than that of the WT during the early stages of fermentation (48-72 hours), but remained significantly higher than the WT during the middle and late stages (72-96 hours), demonstrating a clear difference.

[0076] The pH changes over time during fermentation reveal that, from 48 to 96 hours, the pH of the two strains remained relatively similar, generally within the 4.5-5.0 range, which is consistent with the pH range of the strains during their hormonal production phase. However, between 96 and 120 hours, a significant difference emerged between the two strains: the pH of the WT strain suddenly and rapidly rose to around 6.2, corresponding to the low FK520 production rate of the WT strain in the latter stages. Meanwhile, the pH of the high-yielding strain R2-17-3-10 remained around 5.0, indicating that it was still producing hormonal activity even in the late fermentation phase, which corresponds to the high production rate of R2-17-3-10 in the latter stages. Therefore, the pH trends of the two strains suggest that the pH of the WT strain suddenly rose in the late fermentation phase, exceeding the pH range of hormonal production, while the pH of the R2-17-3-10 strain remained within the hormonal production range.

[0077] The changes in bacterial concentration over time during fermentation reveal similar trends for both strains, with both exhibiting an initial upward and then downward trend. Both strains reached a maximum concentration of approximately 50% at 72 hours, then rapidly declined from 72 to 96 hours, with the rate of decline slowing somewhat between 96 and 120 hours. The only difference was that R2-17-3-10 decreased more rapidly during this period than the WT, ultimately dropping to approximately 30% for R2-17-3-10 and 37% for the WT. Therefore, the differences in bacterial concentration trends between the two strains were not significant, with both reaching a maximum concentration of approximately 50% at 72 hours. However, at the end of fermentation, the concentration of strain R2-17-3-10 was slightly lower than that of the WT.

[0078] The temporal changes in glycerol levels during fermentation revealed that R2-17-3-10 had a higher glycerol level at 48 hours, indicating that it consumed less glycerol during the early growth phase. From 48 to 120 hours, R2-17-3-10 consumed glycerol more slowly than the WT, with the WT maintaining a roughly constant rate of 0.065% / h and R2-17-3-10 maintaining a roughly constant rate of 0.049% / h. At the end of fermentation, the WT had a glycerol concentration of 1.65%, while R2-17-3-10 had a glycerol concentration of 3.55%. Therefore, the glycerol trends indicate that both strains consumed glycerol at a relatively constant rate during the production phase, although the high-yielding strain R2-17-3-10 utilized glycerol more slowly than the WT.

[0079] The temporal changes in amino nitrogen during fermentation revealed that R2-17-3-10 had a higher amino nitrogen level at 48 hours, indicating that it consumed less amino nitrogen during the initial growth phase. Combined with its lower glycerol consumption compared to the WT, R2-17-3-10 maintained a similar bacterial concentration to the WT from 48 to 72 hours, suggesting that the high-yielding strain required fewer nutrients, such as carbon and nitrogen sources, and was therefore more susceptible to growth than the starting strain, the WT. From 48 to 120 hours, both strains showed an increasing trend, with the growth rate steadily increasing, indicating that the production rate of amino nitrogen exceeded the consumption rate in both strains. Furthermore, R2-17-3-10 maintained a consistently higher amino nitrogen level than the WT, likely due to the production of more nitrogenous substances required for FK520 synthesis during the phytochrome production phase, including various acyl-CoA precursors and the enzymes required for precursor synthesis. Therefore, based on the amino nitrogen trend, the high-yielding strain R2-17-3-10 maintained a consistently higher amino nitrogen level than the WT during the phytochrome production phase, potentially facilitating FK520 synthesis and contributing to R2-17-3-10's higher yield.

[0080] The temporal changes in ammonium ion concentrations during fermentation revealed that at 48 h, the ammonium ion concentrations of R2-17-3-10 and WT were 0.357 mmol / L and 0.335 mmol / L, respectively, both below 1 mmol / L. This indicates that both strains had essentially depleted the ammonium sulfate in the culture medium by 48 h, a phenomenon that persisted until 96 h. From 96 to 120 h, the ammonium ion levels of the two strains differed significantly: while both strains increased, the increase in the starting strain WT was greater, reaching a final concentration of 6.215 mmol / L. This may be due to the release of ammonium ions during the later stages of WT's utilization of amino acids as a carbon source, which led to an increase in ammonium ion concentration, corresponding to the sudden and substantial increase in pH. In contrast, the high-yielding strain R2-17-3-10 only saw a slight increase, reaching a final concentration of only 0.709 mmol / L, possibly indicating that lower ammonium ion levels favored the production of FK520. Therefore, judging from the trend of ammonium ion changes, the ammonium ion level of the high-yielding strain R2-17-3-10 was always lower than that of WT during the toxin production stage, which may be more conducive to the synthesis of FK520 and is an important reason for the higher yield of R2-17-3-10.

[0081] Comparison of gene expression levels between R2-17-3-10 and WT

[0082] Total RNA was extracted from the bacterial pellets of the high-yield strain R2-17-3-10 and the starting strain WT at 48h, 72h, and 96h, and then the transcription levels of genes such as fkbN, fkbR2, ​​fkbS, fkbE, fkbO, and fkbL in the FK520 biosynthetic gene cluster were measured by RT-qPCR to analyze the differences between the two at the gene expression level. The results are as follows Figure 11 shown.

[0083] The transcription levels of the fkbR2 gene at different time points show that at 48, 72, and 96 hours, the WT strain exhibited low levels of fkbR2, ​​while the high-yielding strain R2-17-3-10 exhibited significantly higher levels than the WT, with increases of 4.61-fold, 4.92-fold, and 3.59-fold, respectively. Because the fkbR2 gene was overexpressed in strain R2-17-3-10, this suggests that the gene multiplication strategy can achieve increased expression of this gene. While no researchers have specifically studied fkbR2, ​​its function remains unclear. However, the results of this study suggest that it may be a positively regulated gene.

[0084] The transcription levels of the fkbS gene at different time points showed a decreasing trend in both WT and R2-17-3-10. Furthermore, R2-17-3-10 maintained higher levels of fkbS than WT at 48, 72, and 96 hours, increasing by 25%, 27%, and 29%, respectively. This suggests that the regulatory gene fkbR2 may directly or indirectly regulate fkbS transcription. The fkbS gene encodes crotonyl-CoA reductase, which catalyzes the conversion of crotonyl-CoA to ethylmalonyl-CoA. Therefore, increased fkbS expression may help increase the supply of the precursor ethylmalonyl-CoA, thereby improving FK520 production.

[0085] Transcription levels of the fkbE gene at different time points revealed an initial increase followed by a decrease in both WT and R2-17-3-10 expression, reaching their highest levels at 72 hours, with increases of 4.94-fold and 9.10-fold, respectively. R2-17-3-10 showed significant increases compared to WT at 48 and 72 hours, by 3.53-fold and 4.16-fold, respectively, suggesting that the regulatory gene fkbR2 may directly or indirectly regulate fkbE transcription. The fkbE gene encodes a putative homolog of L-carnitine dehydratase, catalyzing the conversion of trihydroxybutyryl-CoA to crotonyl-CoA, ultimately forming ethylmalonyl-CoA. This suggests that increased fkbE expression also promotes the formation of the precursor ethylmalonyl, ultimately promoting the synthesis of FK520.

[0086] The transcription levels of the fkbO gene at different time points showed an increasing trend in the WT, reaching a maximum at 96 hours, 5.50-fold higher than at 48 hours. In contrast, the expression of R2-17-3-10 initially increased and then decreased, with increases of 2.96-fold and 5.62-fold at 48 and 72 hours, respectively, compared to the WT. This suggests that overexpression of the fkbR2 gene in the WT shifts the maximum transcription level of the fkbO gene from 96 hours to 72 hours. This is because fkbO encodes a hydroxyl oxidase, which converts chorismate to DHCHC through multiple reactions. DHCHC, as the initiator unit for the formation of the FK520 macrocycle, plays a crucial role in the synthesis of FK520. This suggests that the regulatory gene fkbR2 may directly or indirectly regulate the transcription of fkbO.

[0087] The transcription levels of the fkbL gene at different time points showed a decreasing trend in both WT and R2-17-3-10. Furthermore, the transcription levels of R2-17-3-10 were consistently higher than those of WT at 48, 72, and 96 hours, increasing by 4.48-fold, 1.18-fold, and 1.00-fold, respectively. This suggests that the regulatory gene fkbR2 may directly or indirectly regulate fkbL transcription. Furthermore, fkbL encodes a lysine cyclodeaminase, which catalyzes the conversion of lysine to pipecolic acid. Pipecolic acid is an elongation unit of NRPS. Under the catalysis of FkbP, pipecolic acid undergoes a condensation reaction with an intermediate synthesized by the PKS, which is an essential step in the formation of the FK520 macrocyclic skeleton. Therefore, increased fkbL transcription may promote the accumulation of the precursor pipecolic acid, ultimately promoting the synthesis of FK520.

[0088] In summary, overexpression of fkbR2 in WT cells increased its own transcriptional level at 48, 72, and 96 hours. Furthermore, fkbN increased by 13.24-fold at 48 hours, suggesting a possible regulatory interaction between fkbR2 and fkbN. Furthermore, the transcriptional levels of fkbE and fkbO, target genes directly regulated by fkbN, also increased at 48 and 72 hours, while fkbL, an indirect fkbN-regulated gene, increased by 4.48-fold at 48 hours. The only gene with a less significant change was fkbS, which also showed an increase of 27%. This suggests that the increased expression of these genes may promote the synthesis of the precursors ethylmalonyl-CoA, DHCHC, and pipecolic acid, ultimately leading to higher R2-17-3-10 production compared to WT. Combined with the changes in the transcription levels of corresponding genes after the combined overexpression of genes fkbS and fkbN in WT, it can be found that overexpression of gene fkbR2 has a greater effect on increasing the expression levels of genes fkbN, fkbE and fkbO, and a smaller effect on increasing the expression levels of genes fkbS and fkbL. This may be the reason why the yield of R2-17-3-10 is lower than that of SN9-11-2-7, and it also shows that genes fkbS and fkbL play an important role in the synthesis of FK520.

[0089] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A Streptomyces hygroscopicus subspecies ascus, characterized in that : Including strain SN9 and strain R2-17.

2. A method for obtaining Streptomyces hygroscopicus subsp. ascus as claimed in claim 1, characterized in that: The regulatory genes fkbN, fkbR1, fkbR2 in the ascomycin biosynthesis gene cluster and the gene fkbS involved in the synthesis of the ascomycin precursor ethylmalonyl-CoA, as well as the regulatory gene SH03686 of the leucine-responsive regulatory protein family (Lrp) outside the gene cluster were overexpressed in Streptomyces hygroscopicus subsp. ascus ATCC 14891 to screen for ascomycin high-producing strains.

3. The method for obtaining Streptomyces hygroscopicus subspecies ascus according to claim 2, characterized in that: Specifically: S1. First, a series of overexpression plasmids are constructed, and then these plasmids are transferred into Streptomyces hygroscopicus subsp. ascus by conjugation to obtain the corresponding ascomycin genetically engineered bacteria; S2. The constructed ascomycin genetically engineered bacteria were then fermented in shake flasks to obtain the strains SN9 and R2-17 with the highest yields; The fermentation processes of S3, strain SN9, strain R2 and the starting strain were compared.

4. The method for obtaining Streptomyces hygroscopicus subspecies ascus according to claim 3, characterized in that: The series of overexpression plasmids include gene fkbS overexpression plasmid pIB139-fkbS, gene fkbS and fkbN combined overexpression plasmid pIB139-fkbSN, gene fkbS and fkbR1 combined overexpression plasmid pIB139-fkbSR1, gene fkbR2 overexpression plasmid pIB139-fkbR2, ​​and gene SH03686 overexpression plasmid pIB139-SH03686.

5. Use of the Streptomyces hygroscopicus subsp. ascus as claimed in any one of claims 1 to 4 for promoting the increase of ascomycin production.

6. The use of Streptomyces hygroscopicus subsp. ascus according to claim 5, characterized in that: After strains SN9 and R2-17 jointly overexpressed the genes fkbS and fkbN and overexpressed the gene fkbR2 in the starting bacteria, the transcription levels of the regulatory genes (fkbN, fkbR2) on the ascomycin biosynthesis gene cluster and the related genes involved in the synthesis of precursors ethylmalonyl-CoA (fkbS, fkbE), pipecolic acid (fkbL), and DHCHC (fkbO) were increased, ultimately promoting the increase in ascomycin production.

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