Natamycin is a polyene macrolide antibiotic with a 26-membered lactone ring, which contains conjugated double bonds. This 26-membered ring is connected to mycosamine at a specific carbon position via an ether linkage, forming a six-membered pyranose ring (Aparicio et al., 2016). As an efficient broad-spectrum antimicrobial agent, natamycin can effectively inhibit the growth of yeasts and molds, with its antimicrobial mechanism varying depending on the species of fungi but always closely related to the action of ergosterol (te Welscher et al., 2008). After comprehensive evaluation by the World Health Organization (WHO), the European Food Safety Authority (EFSA), and Food and Drug Administration (FDA), natamycin has been confirmed as generally recognized as safe (GRAS) substance and classified by the European Union as a natural preservative, assigned the E number (EEC No. 235) (Meena et al., 2021). Its widespread application in food industry, human treatment and agriculture field (Kumar et al., 2021), along with the continuous growth in demand for natamycin in these fields, makes the development of methods to increase its production particularly urgent.

Natamycin, also known as pimaricin, is mainly produced by S. natalensis (Shen et al., 2024), S. chattanoogensis (Liu et al., 2015a), S. gilvosporeus (Liang et al., 2008), and other Streptomyces (Chen et al., 2008). Increasing the natamycin synthesis capacity of the strain is an effective way to reduce its production cost. Non-rational design, including physical mutagenesis, chemical mutagenesis, protoplast fusion, and genome shuffling (Sun et al., 2022a), have been widely employed to develop high-production strains of natamycin. These methods do not require knowledge of the biosynthesis process and regulatory mechanism of natamycin (Wang et al., 2016a). However, strains that have been repeatedly mutated can develop a “fatigue effect” on mutagens. Even if a large number of mutants are obtained, it is difficult to find higher production strains. With the understanding of the synthesis mechanism of natamycin, rational design has been successfully applied to enhance the production of natamycin and deepen our understanding of the regulation of natamycin biosynthesis. In general, the production of natamycin can be enhanced by overexpressing key regulatory genes in its synthesis (Wang et al., 2016b). Rational design mainly involves knocking out or overexpressing specific genes, which requires a clear understanding of metabolic pathways (Bekker et al., 2014). Semi-rational strategy is between rational and non-rational strategy, combining the advantages of both. Utilizing information from known microbial metabolic networks while maintaining a certain degree of randomness. This approach reduces the uncertainty in mutation libraries and increases the success rate of obtaining superior mutants (Chen et al., 2012). The combination of non-rational and semi-rational strategies has shown significant effects in enhancing the production of secondary metabolites (Zhou et al., 2012). Moreover, due to the incomplete understanding of the natamycin metabolic pathway, the rational approach is not as effective. Therefore, the integration of non-rational and semi-rational strategies is the best choice.

In recent years, heavy ion beams have been extensively utilized as a novel source of radiation mutagenesis in plant and microbial breeding, demonstrating promising development prospects. Liu et al. (2018) used heavy ion beams generated at the Heavy Ion Research Facility in Lanzhou to mutagenise Streptomyces fungicidicus and obtained a mutant strain M30 with an enduracidin production of 685.9 mg·L−1, which was 114 % higher than that of the wild-type strain. Song et al. (2017) treated it with a combination of heavy ion beam irradiation and sodium nitrite and obtained a mutant strain S-233 that produced avermectin B1a of 6818 μg·mL−1, which was 23.8 % higher than that of the parental strain. Compared with other mutagenesis techniques, heavy ion beam mutagenesis has a higher linear energy transfer (LET) and relative biological effectiveness (RBE), which can alter the genetic characteristics of biological cells (Hu et al., 2017, Guo et al., 2020a), induce complex DNA damage, and establish a rich phenotype mutant library. Therefore, the aforementioned case demonstrates that heavy ion beam mutagenesis is an effective strategy for enhancing secondary metabolite production in Streptomyces. However, it has not yet been utilized for breeding natamycin-producing bacteria.

Global Transcription Machinery Engineering (gTME) utilizes the unique advantage of multi-point regulation of transcription factors to compensate for the shortcomings of single gene modification in metabolic engineering operations (Chen et al., 2021). It improves the collective phenotypic expression of many genes by changing a single sigma factor (Alper et al., 2006). This technique has been widely used in the selection and breeding of high-production strains and has achieved remarkable results. Xie et al. (2023) used gTME to obtain the highest protease activity of Bacillus pumilus 62 A of 1926 U/mL, which was 2.7 times higher than that of the control strain. Deng et al. (2021) used gTME to increase the Corynebacterium glutamicum S9114 GlcNAc potency to 16.0 g·L−1, which was 92.8 % higher than the control strain. Natamycin biosynthesis is regulated by numerous regulatory factors (Zong et al., 2022). Therefore, the application of gTME technology to mutate natamycin biosynthesis-related regulatory factors may be an effective strategy to enhance the synthesis ability of natamycin.

In this study, we attempted to use a combination of heavy ion beam radiation mutagenesis and gTME technology to enhance the synthesis of S. gilvosporeus. Firstly, a high-throughput cultivation strategy for Streptomyces was established and through the use of heavy ion beam radiation mutagenesis was used to obtain a strain with significantly increased natamycin production. Secondly, the global transcription factor whiG, which has a positive regulatory effect on the biosynthesis of natamycin, was obtained through transcription factor screening. Thirdly, whiG was mutated through gTME, a high-production mutant strain was obtained. This study employed a semi-rational strategy to engineer S. gilvosporeus, achieving a significant increase in natamycin production, and proposing new ideas for enhancing the secondary metabolite production of Streptomyces strains.