Daptomycin is a cyclic lipopeptide antibiotic produced by
To improve the daptomycin productivity, a great deal of effort is committed enhancing the production of daptomycin through strain improvement, which has primarily focused on physical and chemical mutagenesis and genome recombination. Wang et al. (2020) used ultraviolet, microwave, LiCl, and compound mutagenesis to increase daptomycin yield by 20.79% compared with the original strain. Gao et al. (2016) used plasma and UV compound mutagenesis combined with sodium glutamate resistance screening to obtain a high-yield strain and achieved daptomycin production of 3.9 g/l in a 5 l fermentor. Using UV and nitrosoguanidine, Yu et al. (2014) screened eight strains with high daptomycin production to find an initial population for genome shuffling. A high yield strain was screened after the fourth round of fusion and achieved daptomycin production of 38.5 mg/l in a 7.5 l fermentor, which was 3.7-fold higher than the original strain (Yu et al. 2014). Zhang (2021) selected a daptomycin-producing strain by combining UV mutagenesis with NTG mutagenesis and genome rearrangement, and its yield was 1.59-fold higher than that of the original strain.
Compared with the traditional physical and chemical mutagenesis technology, ribosome engineering technology is simple and more effective. It modifies the ribosomal structure of microorganisms via resistance mutation technology so that the regulatory genes related to secondary metabolism are activated directly or indirectly, improving the synthesis ability of secondary metabolites (Ochi et al. 2004; Ochi 2007). This method can be used to increase the yield of secondary metabolites in almost all actinomycetes. More than ten new structural molecules have been discovered from the more than 100 actinomycetes, and the production efficiency of nearly 30 secondary metabolites have been enhanced (Xie et al. 2022). Liu et al. (2021a) produced a new carrimycin high-producing strain RFP40-6-8 using ribosomal engineering technology, with a yield approximately 6-fold higher than the original strain. Lu et al. (2018) used ribosomal engineering technology, and ultraviolet mutagenesis technology to modify
In this study, a genetically stable mutant strain SR-2620 of
The top five mutants with the highest daptomycin production in shake-flask fermentation were chosen as starting strains for the second round of ribosome engineering using 1.6 μg/ml streptomycin. In the third round, 1.8 μg/ml streptomycin was employed, and other conditions were the same as in the first round. The antibiotic concentration was increased by 0.2 μg/ml in each round until the strains no longer produced a sizeable positive mutation.
PCR reaction system: 1 μl of DNA template, 12.5 μl of San Taq PCR Mix enzyme, 1 μl of upstream primer, and 25 μl of downstream primer. PCR amplification system: Step one, pre-denaturation at 95°C for 5 minutes. Step two was denaturation at 95°C for 30 seconds, annealing at 40°C for 30 seconds, extension at 72°C for 30 seconds, for 30 cycles. Step three was extension at 72°C for 10 minutes. The prmier of
The mycelium dry weight was determined as follows: the filter paper was dried at 85°C in the oven to a constant weight, weighed, and set aside. The 10 ml of fermentation broth was centrifuged for 10 min at 12,000 r/min, washed twice with deionized water, filtered on filter paper, dried in the oven at 85°C to a constant weight, and weighed.
The residual sugar was determined by anthrone colorimetry, while the ammonia nitrogen was determined using the reference method (Xie et al. 2005).
Stability experiment on high-yield daptomycin strains.
Strain | First generation of daptomycin production (mg/l) | Second generation of daptomycin production (mg/l) | Third generation of daptomycin production (mg/l) | Fourth generation of daptomycin production (mg/l) | Fifth generation of daptomycin production (mg/l) |
---|---|---|---|---|---|
BNCC 342432 | 10.5 ± 0.2 | 9.9 ± 0.2 | 10.3 ± 0.2 | 9.7 ± 0.2 | 10.4 ± 0.2 |
SR-2009 | 28.5 ± 0.2 | 30.0 ± 0.2 | 24.3 ± 0.2 | 25.0 ± 0.2 | 22.8 ± 0.2 |
SR-2017 | 23.1 ± 0.2 | 21.6 ± 0.2 | 22.8 ± 0.2 | 19.8 ± 0.2 | 14.6 ± 0.2 |
SR-2023 | 29.7 ± 0.2 | 28.8 ± 0.2 | 30.6 ± 0.2 | 29.2 ± 0.2 | 27.2 ± 0.2 |
SR-2026 | 24.2 ± 0.2 | 22.9 ± 0.2 | 21.8 ± 0.2 | 20.1 ± 0.2 | 20.6 ± 0.2 |
SR-2401 | 24.5 ± 0.2 | 25.5 ± 0.2 | 23.4 ± 0.2 | 22.5 ± 0.2 | 24.2 ± 0.2 |
SR-2620 | 38.5 ± 0.2 | 36.9 ± 0.2 | 41.3 ± 0.2 | 36.0 ± 0.2 | 38.5 ± 0.2 |
Ribosomal engineering technology modifies essential components of gene expression, RNA polymerase, and ribosome, to cause mutations in ribosomal-related genes. It then regulates the secondary metabolic pathway of microorganisms, resulting in the overexpression of secondary metabolites. Through transcriptome and metabonomic analysis, Lopatniuk et al. (2019) confirmed that the technology of practical introduction mutations into
The intensity of a round of antibiotic stimulation is relatively low, which can cause heredity instability of the high yield performance of the strain. In this study, a modified method of ribosomal engineering with gradually increasing the stimulation intensity of streptomycin was used to screen for the high-production strain. In this process, the secondary metabolites of the strain were improved. Finally, a streptomycin-resistant mutant SR-2620 with significantly increased daptomycin yield was obtained in a DT plate containing 2.8 μg/ml of streptomycin, while its yield in the shake flask reached 38.5 mg/l. It was 3.7-fold higher than BNCC 342432. In our subsequent studies, we have taken the mutant strain SR-2620 as the starting strain and continued to improve the yield of daptomycin by using the breeding method in this study after changing a new antibiotic. The yield of the new mutant strain has reached 73 mg/l by shaking flask fermentation. Our experimental results prove that this method is feasible and effective. Some studies have demonstrated that the superposition of several antibiotics will further increase the yield of secondary metabolites of the strain (Tamehiro et al. 2003; Wang et al. 2008). In the future study, we will use other new antibiotics to apply continuous resistance stimulation to the strain to improve the yield of daptomycin further.
In this study, a mutant strain SR-2620 was screened by modified ribosome engineering and achieved daptomycin production of 38.5 mg/l in shake-flask fermentation. However, the shaking flask fermentation makes it impossible to control the nitrogen source, carbon source, dissolved oxygen, and pH during the fermentation process. Zhou and Zhang (2018) used a 100 l fermentor feeding experiment to reach the daptomycin titer of 2,276 mg/l. Liu et al. (2019b) optimized the fermentation process using the response surface method and increased daptomycin production by 132%. Therefore, we will carry out a fermentation tank experiment and optimize fermentation conditions to further improve the yield of daptomycin in future research.