Butenyl-spinosyn, a type I polyketide macrolide compound [1], [2], is derived from spinosyns produced through the aerobic fermentation of Saccharopolyspora pogona. It targets the nicotinic acetylcholine receptor (nAChR) and the γ-aminobutyric acid (GABA) receptor in insects, disrupting their nervous systems. This leads to impaired muscle contractions and exhaustion [3], resulting in rapid contact insecticidal action. Compared to its structural analog, spinosad, butenyl-spinosyn displays a broader insecticidal spectrum, effectively controlling pests such as Lepidoptera, Thysanoptera, Coleoptera, Hymenoptera, and Diptera. It is considered a promising green biopesticide with substantial development potential [4], [5]. However, the current production yield of butenyl-spinosyn from wild-type S. pogona remains exceedingly low. Short-chain acyl-CoAs such as acetyl-CoA, propionyl-CoA, malonyl-CoA and methylmalonyl-CoA often serve as the initiation and extension units of polyketide biosynthesis, and play a central role in carbon chain extension during their synthesis[6]. butenyl-spinosyn biosynthesis can be divided into two main stages: biosynthesis and cyclization of the polyketide chain, and glycosylation and methylation modification of the cyclized product. The strategy of precursor metabolic engineering is a useful strategy for constructing high-yield engineering strains of polyketide natural products. An et al. [7] increased acyl-CoA precursor supply by overexpressing the genes for acetyl-CoA carboxylase, propionyl-CoA carboxylase, and acetyl-CoA synthetase in a heterologous polyketide producer, Streptomyces albicans J1074, which led to an increase in polyketide production, suggesting that enhanced precursor supply is important for increasing antibiotic production. Strategies to enhance S. pogona yields include mutation breeding [8], high-yield strain screening [9], ribosome engineering [10], and medium optimization. While traditional strain breeding can increase butenyl-spinosyn yield to a degree, it is prone to mutation reversal, and the extensive workload associated with strain screening, complexity in medium composition, and elevated production costs continue to challenge efficiency improvement. Omics technologies have advanced the study of natural product biosynthesis, enabling rapid genetic information mining [11]. In conjunction with actinomycete genome analysis techniques, significant enhancements in secondary metabolite production have been achieved through structural gene overexpression [12], genome simplification [13], and promoter engineering using gene editing technologies [14], [15]. Genetic modification of the genome by means of histological techniques and molecular biology enables rapid determination of the role of proteins in target strains [16]. Rang et al. [17] performed a comparative proteomic and metabolomic analysis between S. pogona and S. spinosa, identifying a substantial reduction in the overall abundance of proteins involved in butenyl-spinosyn biosynthesis. By overexpressing genes related to rhamnose synthesis (gtt, gdh, epi, k re ) and the methionine adenosyltransferase gene metK, they achieved an impressive 2.7-fold and 3.0-fold increase in production, respectively. Therefore, detailed investigation into the metabolic network of butenyl-spinosyn biosynthesis, targeted genetic modifications, and the identification of key regulatory proteins are crucial for enhancing butenyl-spinosyn production. Currently, a variety of DNA editing technologies, including the Red/ET recombination system, Cre/loxP system, Flp/FRT system, Latour system, and CRISPR system [18], [19], [20], [21], [22], [23], have been developed for microbial genome manipulation. Compared to model organisms such as S. cerevisiae and E. coli, genetic manipulation tools for Streptomyces are relatively scarce. The CRISPR/Cas9 gene editing system, however, provides a versatile means for deletion, introduction, and site-directed mutagenesis of target genes without necessitating exogenous gene insertion. Additionally, controlling temperature conditions can facilitate the removal of the corresponding vector [24]. This technique has been successfully applied to various microorganisms, including E. coli, Bacillus subtilis, Streptomyces, and yeast [25], [26], [27], [28], offering a novel technical strategy for the genetic manipulation of S. pogona.

Cold shock proteins (CSPs) are a ubiquitous class of proteins found in microorganisms, characterized by a molecular weight of approximately 7 kDa. They are rapidly inducible under low-temperature conditions. Serving as molecular chaperones for RNA molecules, CSPs play a pivotal role in vivo by facilitating the folding process of most RNA. This process is aided by various chaperone proteins that regulate the structure and function of RNA, assisting in folding, modification, transport, degradation, and promoting the attainment of the most thermodynamically favorable conformation by reducing energy barriers between competing conformations [29]. CSPs are found across different species, constituting a small yet highly conserved protein family [30]. Despite the wide variety of microorganisms and the variations in CSP types, coupled with their complex mechanisms of action, our understanding of CSP functions remains somewhat limited [31]. Previous studies have shown that S. coelicolor, akin to other bacteria, possesses a conserved family of Csp homologs, indicating a widespread presence among Streptomyces species. Whether CSPs play a key role in regulating the production of secondary metabolites remains to be seen; however, the data provided strongly suggest their potential involvement [32]. CspA, one of the earliest proteins induced during the cold shock response, binds to single-stranded nucleic acids, maintaining the linear structure of mRNA secondary structure and stabilizing mRNA molecules. This activity facilitates a smooth translation process and effectively regulates gene expression [33]. Beyond merely disrupting the secondary structure of mRNA to aid protein translation, CspA employs an additional mechanism to enhance its own translational efficiency, thereby optimizing its functional role even in the absence of cold-induced specific ribosome factors, such as CsdA and RbfA [34]. Notably, CspA is not strictly classified as a "cold shock" protein; it can also be induced by nutrient addition or dilution into the E. coli culture medium[35], along with antibiotic supplementation[36], [37]. This indicates that CspA expression enhances cellular adaptation to adverse external conditions. The genome of Streptomyces pogona encodes ten putative CSP homologs. While the csp gene family has been extensively studied in model organisms such as E. coli, where phenotypic effects are typically observed only upon deletion of three or more paralogs [38], the specific roles of individual csp in actinomycetes remain poorly understood.

In this study, the cold shock protein gene cspA (SP_2649) in S. pogona NRRL 30141 was subjected to genetic manipulation for the first time, employing metabolic engineering techniques that included both deletion and overexpression strategies. Preliminary findings suggest that cspA fulfills essential biological roles in S. pogona NRRL 30141. Furthermore, the effects of CspA protein overexpression on the metabolic pathway modifications in S. pogona NRRL 30141 were elucidated through comparative proteomics and targeted metabolomics analyses. This research is anticipated to uncover the regulatory mechanisms influencing metabolic pathway alterations at a molecular level.