SenX3-RegX3, an Important Two-Component System, Regulates Strain Growth and Butenyl-spinosyn Biosynthesis in Saccharopolyspora pogona.

Butenyl-spinosyn produced by Saccharopolyspora pogona exhibits strong insecticidal activity and a broad pesticidal spectrum. Currently, important functional genes involved in butenyl-spinosyn biosynthesis remain unknown, which leads to difficulty in efficient understanding of its regulatory mechanism and improving its production by metabolic engineering. Here, we present data supporting a role of the SenX3-RegX3 system in regulating the butenyl-spinosyn biosynthesis. EMSAs and qRT-PCR demonstrated that RegX3 positively controls butenyl-spinosyn production in an indirect way. Integrated proteomic and metabolomic analysis, regX3 deletion not only strengthens the basal metabolic ability of S. pogona in the mid-growth phase but also promotes the flow of the acetyl-CoA produced via key metabolic pathways into the TCA cycle rather than the butenyl-spinosyn biosynthetic pathway, which ultimately leads to continued growth but reduced butenyl-spinosyn production. The strategy demonstrated here may be valuable for revealing the regulatory role of the SenX3-RegX3 system in the biosynthesis of other natural products.

Introduction

Butenyl-spinosyn is a secondary metabolite produced by Saccharopolyspora pogona (S. pogona) (Paul et al., 2009; Huang, et al., 2009). It is known to be an excellent insecticidal agent with good potency and a broad spectrum of activity against a variety of Lepidoptera, Diptera, and Coleoptera and a low level of side effects on the non-target organism. At present, butenyl-spinosyn biosynthetic gene cluster (bus cluster) and its biosynthetic pathway have been elaborated (Hahn et al., 2006). In addition, the regulatory mechanism of butenyl-spinosyn biosynthesis has been preliminarily studied. Although no key regulatory genes were found in the 110-kb bus cluster, pnp and afsR were identified outside the bus cluster to have significant effects on the butenyl-spinosyn biosynthesis (Li et al., 2018, 2019). Despite these findings, our knowledge of the complex regulatory network underlying butenyl-spinosyn biosynthesis is still limited and fragmentary, which presents an obstacle to rational design of butenyl-spinosyn high-producing strains through metabolic engineering.

The biosynthesis of natural products produced by most actinomycetes is affected by phosphate concentrations in the medium (Romero-Rodríguez et al., 2018; Martín et al., 2012; Solans et al., 2019). Initially, the phosphate effect was attributed to a decrease or inhibition of natural product production as a result of a phosphate increase in the culture medium (Curdová et al., 1976). Further studies on phosphate metabolism-related genes identified several of these genes, such as alkaline phosphatase, polyphosphatase, and low- and high-affinity transporter genes, and the pho regulon (Moura et al., 2001; Vuppada et al., 2018; Santos-Beneit et al., 2008; Martínez-Castro et al., 2018). Additionally, it was found that the molecular mechanism of phosphate control is mainly regulated by a two-component system (Martín et al., 2017).

Currently, there are two kinds of two-component systems known to be involved in phosphate regulation, the PhoR-PhoP system and the SenX3-RegX3 system (Aggarwal et al., 2017; White et al., 2018). The PhoR-PhoP system is conserved in all Streptomyces and most other sequenced actinomycetes (Barreiro and Martínez-Castro, 2019). Many studies have demonstrated that this system plays an important regulatory role in cellular differentiation and secondary metabolite biosynthesis in actinomycetes (Sola-Landa et al., 2003, 2005, 2013; Martín et al., 2019). In another system that senses and responds to phosphate, the sensor kinase SenX3 and the response regulator RegX3 were first identified and have been well studied in Mycobacterium (Namugenyi et al., 2017; Rifat et al., 2014; Park et al., 2019). In Mycobacterium smegmatis (M. smegmatis), SenX3-RegX3 has been shown to control the expression of phosphate-dependent genes such as those of the pstSCAB and phnDCE operons and is required for optimal growth under Pi-limiting conditions (Gebhard and Cook, 2008). Meanwhile, SenX3-RegX3 plays an important role in regulating M. tuberculosis membrane vesicle production (White et al., 2018). Despite these characteristics, the function of this system in the regulation of natural product biosynthesis has not been reported. A genomic analysis of S. pogona showed that it harbored a complete SenX3-RegX3 system and can be used as a good model for studying the interaction effects between the SenX3-RegX3 system and the regulation of strain growth development and natural product production.

In the present study, we constructed regX3 deletion and overexpression mutants and investigated the effect of RegX3 on the changes in the strains phenotypes. The regX3 deletion mutant was used to determine the regulatory mechanism of RegX3-related butenyl-spinosyn biosynthesis at the proteome and metabolome levels and further demonstrated that RegX3 could control target product production by regulating the primary metabolism and the expression of the bus cluster. Using electrophoretic mobility shift assays (EMSAs), we found that RegX3 could not bind to the bus cluster promoter, suggesting that RegX3 indirectly activates the expression of the bus cluster. This work deepens our understanding of butenyl-spinosyn regulatory mechanisms and lays an important foundation for studying the regulatory mechanism of the SenX3-RegX3 system in natural product biosynthesis in other actinomycetes.

Results

Effect of Phosphate on Butenyl-spinosyn Production and Strain Growth

Given the paucity of data regarding the effect of inorganic phosphate (Pi) on S. pogona butenyl-spinosyn biosynthesis, we measured its production under the following conditions: Pi-free, low Pi concentration (2.5 μM), and high Pi concentration (4 mM). The results of HPLC, bioassay, and LC-MS/MS confirmed that the chromatographic peak found at 10.67 min was butenyl-spinosyn (Figures 1A, S1, and S2). Next, this chromatographic peak was used as a basis for comparing the butenyl-spinosyn production changes. The results showed that the addition of increasing concentrations of Pi drastically reduced butenyl-spinosyn production in synthetic fermentation medium (SFM) (Figure 1A). Moreover, the promoters that control the expression of polyketide synthase genes (busA, busB, busC, busD, busE) are subject to phosphate modulation (Figure 1B). Meanwhile, the strain density in the stationary period was decreased under a higher Pi concentration at the same sampling time (Figure 1C). It was demonstrated that butenyl-spinosyn biosynthesis and strain growth are highly sensitive to Pi control.

Figure 1

Effect of Phosphate on Butenyl-Spinosyn Production and Strain Growth

(A) Effect of increasing Pi concentrations (0, 2.5 × 10−3, and 4 mM) on butenyl-spinosyn production.

(B) Effect of increasing Pi concentrations (0, 2.5 × 10−3, and 4 mM) on the gene expression of butenyl-spinosyn polyketide synthases. S. pogona was inoculated into SFM with different Pi concentrations and cultured at 30°C for 4 days. Total RNA was then isolated and used for qRT-PCR assays. The control was free Pi. The 16S rRNA served as the normalization control (∗∗p < 0.01; ∗p < 0.05; n = 4).

(C) Effect of increasing Pi concentrations (0, 2.5 × 10−3, and 4 mM) on strain growth.

Since the regulatory mechanism of Pi is controlled by the SenX3-RegX3 system, we are interested in the role of this system in S. pogona (Figures S3 and S4). To assess the in vivo biological significance of RegX3, we constructed regX3 deletion and over-expression mutants as well as the complemented strain (Table 1, Figures S5 and S6).

Table 1

Strains and Plasmids in This Study

Strains/Plasmids	Description	Source
S. pogona
NRRL 30141	S. pogona, wild-type strain, plasmid-free parental strain	This lab
S. pogona-ΔregX3	S. pogona, regX3 deletion strain	This work
S. pogona::regX3	S. pogona, regX3 over-expressed strain	This work
S. pogona-ΔregX3::regX3	S. pogona-ΔregX3, regX3 complemented strain	This work
E. coli
DH5α	Escherichia coli, plasmid-free, host for general cloning	This lab
BL21	Escherichia coli, plasmid-free, host for heterologous protein expression	This lab
Plasmids
pOJ260	E. coli-cloning vector, containing pUC18 replicon, oriT, AprR	This lab
pKC-tsr	acc(3)IV, pSG5, j23119, vector for expression	This lab
pOJ260-cm-PermE	pOJ260 containing PermE, for PermE PCR amplification	This lab
pOJ260-UAregX3-apr-DAregX3	pOJ260 containing homologous region flanking regX3, for regX3 deletion	This work
pOJ260-PermE-regX3	pOJ260 containing PermE and regX3, for regX3 overexpression	This work
pKC-tsr-regX3	pKC-tsr containing regX3, for regX3 complementation	This work

RegX3 Regulates polyP Accumulation via PPK-Dependent mprAB-sigE-rel Signaling Cascade in the Intermediate Growth Stage

Inorganic polyphosphate (polyP) is a linear biopolymer of phosphoanhydride-linked phosphate units that is found in all domains of life (Barreiro and Martínez-Castro, 2019). In diverse bacteria, polyP is essential for the stress response, motility, biofilm formation, cell cycle control, natural biosynthesis, and virulence (Esnault et al., 2017; Rao et al., 2009). We examined whether changes in the expression level of the S. pogona regX3 gene altered polyP storage during fermentation (Figure 2A). It was found that the S. pogona-ΔregX3 and S. pogona::regX3 mutants showed no significant change in polyP accumulation compared with the original strain during early development. However, intracellular polyP was obviously decreased in the S. pogona-ΔregX3 mutant but increased in the S. pogona::regX3 mutant in the intermediate growth stage. The complemented strain S. pogona-ΔregX3::regX3 restored the original polyP level, suggesting that S. pogona RegX3 can positively regulate polyP accumulation. It has been reported that polyP is synthesized by polyphosphate kinase (PPK) and establishes a PPK-dependent mprAB-sigE-rel signaling cascade linking polyP metabolism to the stringent response in M. tuberculosis (Liang et al., 2017; Kulakovskaya and Kulaev, 2013; Sureka et al., 2007). The result of qRT-PCR showed that the transcriptional level of ppk was significantly downregulated in the S. pogona-ΔregX3 mutant, whereas the opposite pattern was observed in the S. pogona::regX3 mutant, indicating that RegX3 also regulates polyP accumulation by the PPK-dependent mprAB-sigE-rel signaling cascade in S. pogona (Figure 2B).

Figure 2

Effect of regX3 Deletion and Overexpression on Polyphosphate (polyP) Accumulation

(A) Comparative analysis of polyP accumulation in different strains. Wild-type S. pogona, S. pogona-ΔregX, and S. pogona::regX3 mutants and complemented strain S. pogona-ΔregX::regX3 were inoculated into SFM at equal OD600 values and grown at 30°C. Cells in the early and intermediate stages of growth were collected, and their polyP contents were determined via the DAPI method. The data shown are the mean values ± standard deviations of three independent experiments (∗∗p < 0.01; ∗p < 0.05; ns, no significance; n = 3).

(B) The ppk transcript was measured in different strains. The cells of the different strains were inoculated into SFM and cultured at 30°C for 4 days. Total RNA was then isolated and used for qRT-PCR assays. The control strain was wild-type S. pogona. 16S rRNA served as the normalization control (∗∗p < 0.01; ns, no significance; n = 4).

Effects of RegX3 Expression Abundance Changes on S. pogona Butenyl-spinosyn Biosynthesis and Other Physiological and Biochemical Characteristics

To determine the roles of RegX3 in butenyl-spinosyn biosynthesis, HPLC was used to estimate butenyl-spinosyn production by the S. pogona-ΔregX3 and S. pogona::regX3 mutants. The peak areas of butenyl-spinosyn in the original strain and the S. pogona-ΔregX3 and S. pogona::regX3 mutants were 1,084.3 ± 72.37, 305.87 ± 32.02, and 682.57 ± 58.60 mAU∗s, respectively (Figures 3A–3C). Meanwhile, the complemented strain S. pogona-ΔregX3::regX3 restored the original butenyl-spinosyn level (Figure S7). Therefore, regX3 deletion significantly decreased the production of butenyl-spinosyn by 71.8% compared with that in the original strain. Surprisingly, regX3 overexpression also decreased the production of butenyl-spinosyn by 37.05% compared with that in the original strain. The above polyP analysis results showed that the overexpression of regX3 greatly promoted the absorption of extracellular Pi by S. pogona and resulted in polyP overproduction, which may be an important reason for the decrease in butenyl-spinosyn production. To confirm this hypothesis, we estimated the butenyl-spinosyn production of the S. pogona::regX3 mutant in the SFM without Pi and found that its production was increased by 230% compared with that in SFM (Figure 3D). Moreover, the qRT-PCR results showed that regX3 deletion resulted in a significant decrease in the expression of busA, busF, busG, and busI (Figures 3E and 3F). As expected, regX3 overexpression in SFM also led to the downregulation of the expression of these genes, but the expression of busA, busF, busG, and busI was significantly upregulated in the SFM without Pi. To examine whether phosphorylated RegX3 might directly regulate transcription of the bus cluster, His6SenX3 and His6RegX3 were expressed in E. coli BL21, and their affinity toward the three regions containing bus promoters was examined in EMSAs (Figure 3G). The results showed that RegX3 did not bind to the bus promoters, demonstrating that RegX3 may regulate butenyl-spinosyn production by indirectly promoting the expression of the bus cluster (Figure 3H).

Figure 3

RegX3 Positively Regulates Butenyl-Spinosyn Production in S. pogona

(A–C) Comparison of butenyl-spinosyn production in wild-type S. pogona and the S. pogona-ΔregX and S. pogona::regX3 mutants in SFM via HPLC analysis.

(C and D) Comparison of butenyl-spinosyn production in the S. pogona::regX3 mutant in SFM and SFM without Pi via HPLC analysis.

(E) Genetic organization of the bus cluster in S. pogona.

(F) Effect of regX3 deletion and overexpression on the transcription level of bus cluster in SFM or SFM without Pi. The cells of the different strains were inoculated into SFM or SFM without Pi and cultured at 30°C for 4 days. Total RNA was then isolated and used for qRT-PCR assays. The control strain was wild-type S. pogona. 16S rRNA served as the normalization control (∗∗p < 0.01; ∗p < 0.05; ns, no significance; n = 4).

(G) Heterologous expression of regX3 and senX3. Lane M, protein marker; lane 1 and 2, heterologous expression of regX3 in E. coli BL21; lane 3–5, heterologous expression of senX3 in E. coli BL21; lane 6, E. coli BL21 (Control).

(H) EMSAs assessing the interaction of the P, P, and P probes with the purified His6-tagged RegX3-P protein.

For other physiological and biochemical characteristics, the S. pogona-ΔregX3 mutant exhibited a slower growth rate and glucose consumption rate compared with the original strain during the test period (Figure 4). However, the S. pogona::regX3 mutant also showed inhibited strain growth and glucose consumption, and its inhibition was slightly greater than that of the S. pogona-ΔregX3 mutant, indicating that RegX3 plays an important role in maintaining the normal growth development of S. pogona.

Figure 4

Effect of regX3 Deletion and Overexpression on Strain Growth Kinetics and Glucose Consumption

(A) Growth kinetics analysis of wild-type S. pogona and its derivatives in SFM.

(B) Comparison of glucose utilization in wild-type S. pogona and its derivatives in SFM.

Overview of the Comparative Proteomic and Targeted Metabolomic Analysis

To explain why regX3 deletion causes the above phenomenon, we further carried out a comparative proteomics analysis using iTRAQ labeling in the S. pogona-ΔregX3 mutant. According to the butenyl-spinosyn production analysis, the butenyl-spinosyn production of the S. pogona-ΔregX3 mutant began to stabilize on day 6 (Figure S8). This time point was selected for protein extraction from the S. pogona-ΔregX3 mutant and original strain separately. The resulting mass spectra were searched against the S. pogona proteome database, and a total of 2,533 proteins were identified (unique peptides >1) (Data S1, Figure 5A). Using a 1.33-fold cutoff, there were 128 proteins differentially expressed between the S. pogona-ΔregX3 mutant and original strain (Data S2, Figure 5B). These differentially expressed proteins were involved in multiple biological processes, such as glycolysis, pentose phosphate pathway (PP pathway), TCA cycle, fatty acid metabolism, oxidative phosphorylation, amino acid metabolism, two-component system, and ABC transporters, which suggests that RegX3 is likely to be a global regulator.

Figure 5

Comparative Proteomic Analysis between the S. pogona-ΔregX Mutant and Wild-Type S. pogona.

(A) Venn diagram of protein identification in two biological replicates. The number of proteins is shown in each area.

(B) KEGG pathway analysis of differentially expressed proteins.

To analyze whether the changes in metabolite abundance related to strain growth and target product biosynthesis were consistent with the changes in proteins, the intracellular metabolome was evaluated by LC-MS/MS. The samples from day 6 were also used for metabolomic profiling to analyze the correlation with the proteomic data. As a result, 21 intracellular metabolites were identified and quantified by LC-MS/MS, which are mainly involved in glycolysis, PP pathway, TCA cycle, and oxidative phosphorylation (Table 2, Data S3).

Table 2

regX3 Deletion Resulted in Differential Expression of Metabolites Related to Energy Metabolism

Metabolites	KEGG Pathway	Fold change (S. pogona-ΔregX3/S. pogona)	p Value
Succinate	TCA cycle	2.813	0.002
Beta-D-Fructose 6-phosphate	Glycolysis	2.492	0.017
NADH	Oxidative phosphorylation	2.093	0.001
L-Malic acid	TCA cycle	1.932	0.018
D-Glucose 6-phosphate	Glycolysis	1.741	0.014
NAD	Oxidative phosphorylation	1.685	0.011
Citrate	TCA cycle	1.678	0.004
Alpha-ketoglutarate	TCA cycle	1.595	0.030
NADP	Pentose-phosphate pathway	1.526	0.043
Pyruvate	Glycolysis	0.410	0.004
Lactate	Glycolysis	0.258	0.025
Fumarate	TCA cycle	0.249	0.026
AMP	Oxidative phosphorylation	0.169	0.006
Phosphoenolpyruvate	Glycolysis	0.101	0.004
FMN	Oxidative phosphorylation	0.098	0.004

RegX3 Deletion Leads to an Increased Acetyl-CoA Metabolic Flux to the TCA Cycle

The current study provides an overview of the central carbon metabolic pathway and the changes in the expression levels of proteins and metabolites involved in key metabolic pathways, as shown in Figure 6. Most enzymes and their catalytic products involved in central carbon metabolism could be identified from the shotgun proteomic and targeted metabolomic data. PTS glucose transporter IIA (PTS IIA) was identified, and its abundance in the S. pogona-ΔregX3 mutant was higher than that in the original strain. As shown in the metabolomic analysis, glucose 6-phosphate presented higher abundance in the S. pogona-ΔregX3 mutant, indicating that the ability of the S. pogona-ΔregX3 mutant to transport and phosphorylate glucose was stronger than that of the original strain, which was consistent with the rapid glucose consumption rate and the improvement of cell growth.

Figure 6

Effect of regX3 Deletion on Primary Metabolism in S. pogona.

This regulatory network is based on the analysis of differentially expressed proteins and metabolites associated with glucose transport, glycolysis, the pentose phosphate pathway, the TCA cycle, and fatty acid degradation. Reactions are reported according to KEGG metabolic pathway databases. Triangle represents a change in protein abundance, and arrow represents a change in metabolite abundance.

Glycolysis and the PP pathway are two main mechanisms whereby microorganisms metabolize glucose (Seol et al., 2016; Nikel et al., 2015). Throughout glycolysis, the abundance of 6-phosphofructokinase (pfkA), which is a rate-limiting enzyme for glycolysis, and its catalytic product fructose 1,6-diphosphate in the S. pogona-ΔregX3 mutant was higher than that in the original strain. However, other enzymes (phosphoglycerate mutase [gpmA], enolase [eno], and pyruvate dehydrogenase [aceE, bkdC1]) and metabolites (phosphoenolpyruvate and pyruvate) involved in glycolysis, their abundance in the S. pogona-ΔregX3 mutant was opposite. The PP pathway is the second most important pathway for glucose metabolism, and its initial substrate is glucose 6-phosphate (Nikel et al., 2015). We found that no differentially expressed proteins related to the oxidative branch of the PP pathway were detectable in the S. pogona-ΔregX3 mutant. Interestingly, differentially expressed proteins (transketolase [tktA2], transaldolase [tal], and ribulose-phosphate 3-epimerase [rpe]) and fructose 6-phosphate related to the non-oxidative branch of the PP pathway were identified in the S. pogona-ΔregX3 mutant, and their abundance was significantly higher than in the original strain. In addition, ribose-phosphate pyrophosphokinase (prsA) was identified in our proteomic data, which is one of the rate-limiting enzymes of purine metabolism, and its abundance was also higher than in the original strain. The above detection results suggest that regX3 inactivation can improve the abilities of glycolysis, compounds with different carbon numbers to undergo mutual conversion and synthesize ATP, GTP, and their derivatives, but inhibit the generation of acetyl-CoA from pyruvate.

Fatty acid degradation metabolism is an important metabolic pathway for the synthesis of acetyl-CoA (Kallscheuer et al., 2017). The proteomic data obtained in the present study showed that the abundance of enoyl-CoA hydratase (echA), 3-hydroxybutyryl-CoA dehydrogenase (fadB), and acetyl-CoA acyltransferase (fadA, catF) in the S. pogona-ΔregX3 mutant was higher than that in the original strain. These data indicate that RegX3 inactivation increased the degradation ability of fatty acid. The acetyl-CoA produced through glycolysis and fatty acid degradation metabolism will enter the TCA cycle to produce energy and several metabolic precursors for cell growth and the synthesis of other metabolites (Vuoristo et al., 2017). Citrate synthase (citA), isocitrate dehydrogenase (icd-2), and 2-oxoglutarate dehydrogenase (sucB) are the rate-limiting enzymes of the TCA cycle and were detected in our proteomic data. The abundance of these three rate-limiting enzymes and other enzymes involved in the TCA cycle, such as aconitate hydratase (acn), succinyl-CoA synthetase (sucC, sucD), fumarate reductase (fumA), and malate dehydrogenase (mdh), in the S. pogona-ΔregX3 mutant was higher than that in the original strain. Additionally, citrate, 2-oxoglutarate, succinate, and malate exhibited higher abundance in the S. pogona-ΔregX3 mutant, although the abundance of succinate dehydrogenase (sdhA) and its catalytic product fumarate was low. These data suggest that the S. pogona-ΔregX3 mutant shows a greater metabolic capacity than the original strain in terms of central carbon metabolism, so it can continue to absorb and utilize nutrients in the extracellular environment during the analysis phase. Moreover, the metabolic flux flowing through the TCA cycle in the S. pogona-ΔregX3 mutant is increased compared with that in the original strain, which may increase the flow of the acetyl-CoA produced via glycolysis, fatty acid degradation, and other metabolic pathways into the TCA cycle and ultimately limits butenyl-spinosyn biosynthesis.

Discussion

The effect of inorganic phosphate on the physiology and antibiotic production of most actinomycetes has been known for some time (Romero-Rodríguez et al., 2018; Barreiro and Martínez-Castro, 2019). In general, it has been observed that high phosphate concentrations in the culture medium stimulate strain growth and inhibit natural product biosynthesis, as occurs in S. filipinensis (filipin), S. avermitilis (avermectin), and S. clavuligerus (clavulanic acid) (Martín et al., 2017; Barreales et al., 2018). In our study, phosphate also affects growth development and butenyl-spinosyn biosynthesis in S. pogona. Three phosphate systems with different concentrations (0, 2.5 × 10−3, and 4 mM) were established in this study; however, the bacterial density and target product production in the medium without phosphate were significantly higher than those in the low-phosphate concentration and high-phosphate concentration systems. These results demonstrate that butenyl-spinosyn production is also highly sensitive to phosphate control, in contrast to the lower sensitivity of other classes of secondary metabolites, such as orthosomycin antibiotics (Zhu et al., 2007) or cephalosporins (Leite et al., 2016), that are only inhibited at high phosphate concentrations (20–100 mM).

Recent studies have shown that the absorption and assimilation of extracellular phosphate by actinomycetes is related to the regulation of the intracellular Pho regulon, whereas the pho regulon is usually controlled by two-component systems (Sola-Landa et al., 2013; Allenby et al., 2012; Glover et al., 2007). PhoR-PhoP and SenX3-RegX3 are two classes of two-component systems associated with phosphate regulation. Although the PhoR-PhoP and SenX3-RegX3 systems are related to phosphate regulation, only the PhoR-PhoP system has been reported in Streptomyces, and it negatively regulates the biosynthesis of most natural products (Sola-Landa et al., 2003; Barreiro and Martínez-Castro, 2019; Yang et al., 2015; Mendes et al., 2007). The analysis of sequenced Streptomyces genomes showed that most Streptomyces do not contain the senX3-regX3 system, with the exception of S. coelicolor, S. griseoruber, and S. davaonensis. Therefore, no research on the regulatory mechanism of the SenX3-RegX3 system related to natural product biosynthesis has been reported. However, the SenX3-RegX3 system is found in the genomes of various Mycobacterial species, indicating that this evolutionarily conserved two-component system may play a fundamental regulatory role in Mycobacterial physiology, such as bacterial survival and chronic infections in mouse lungs (Rifat et al., 2014; James et al., 2012). The genomic analysis of S. pogona showed that it exhibited a complete senX3-regX3 system and the organization of senX3-regX3 and the adjacent gene gpmA and ppx-gppA was the same as in the homologous region in M. smegmatis, suggesting that the senX3-regX3 system in S. pogona may present similar transcription patterns and biological functions to those of M. smegmatis (Figure S9). Pi has an important influence on strain growth and natural product biosynthesis, and its regulatory mechanism can be controlled by the SenX3-RegX3 system (Glover et al., 2007). Therefore, this system is likely to exhibit a special relationship with the normal growth development and butenyl-spinosyn biosynthesis of S. pogona.

In this work, the inactivation of regX3 obviously decreased the production of butenyl-spinosyn by 71.8%. Moreover, the expression levels of some bus genes in the S. pogona-ΔregX3 mutant were significantly downregulated compared with those in the original strain. EMSA showed that the SenX3-RegX3 system regulates butenyl-spinosyn biosynthesis by indirectly promoting the expression of the bus cluster. These data suggest that RegX3 may act as a pleiotropic regulator to control butenyl-spinosyn biosynthesis. Proteomic and targeted metabolomic analyses were first used to compare the S. pogona-ΔregX3 mutant and original strain. A total of 55 proteins and 10 metabolites were found to be upregulated, whereas 63 proteins and 6 metabolites were downregulated in the S. pogona-ΔregX3 mutant. These differentially expressed proteins are involved in multiple biological processes such as glycolysis, PP pathway, TCA cycle, fatty acid metabolism, oxidative phosphorylation, amino acid metabolism, two-component system, and ABC transporters, which further explains the regulatory characteristics of RegX3.

Acetyl-CoA, malonyl-CoA, methylmalonyl-CoA, rhamnose, forosamine, and S-adenosyl methionine are important precursors for butenyl-spinosyn biosynthesis (Huang et al., 2009), which are mainly produced through primary metabolic activities. The proteome and metabolome data allowed the identification of most proteins and metabolites involved in the primary metabolism, among which the upregulated proteins not only promote the conversion of extracellular glucose to phosphorylated glucose but also enhance the fatty acid degradation ability and the nonoxidative PP pathway, which provides sufficient precursors for other biological processes. The TCA cycle is a central pathway for the metabolism of carbon sources, lipids, and amino acids and represents a major energy source for cells (Vuoristo et al., 2017). In the S. pogona-ΔregX3 mutant, most proteins involved in the TCA cycle are upregulated, including aconitate hydratase, isocitrate dehydrogenase, 2-oxoglutarate dehydrogenase, succinyl-CoA synthetase, fumarate reductase, and malate dehydrogenase. Therefore, the proportion of acetyl-CoA produced through glycolysis, fatty acid degradation, and other metabolic activities entering the TCA cycle is increased, whereas the amount of acetyl-CoA utilized for butenyl-spinosyn biosynthesis is reduced. These results may explain why the S. pogona-ΔregX3 mutant exhibited low butenyl-spinosyn production and increased biomass.

In conclusion, we have shown that the SenX3-RegX3 system plays a very important role in the regulation of growth development and butenyl-spinosyn biosynthesis in S. pogona. Our present findings provide a strategy for the improvement of butenyl-spinosyn production based on the engineering of regX3. Although multiple differentially expressed proteins affected by RegX3 were identified based on comparative proteomic analysis, the detailed regulatory mechanism is not clear. Therefore, chromatin immunoprecipitation sequencing (ChIP-seq) analysis and in vitro characterization of RegX3 targets will be useful for further clarifying the regulatory role of RegX3 in butenyl-spinosyn biosynthesis in S. pogona.

Limitations of the Study

In this study, we provide a systematic perspective to demonstrate the SenX3/RegX3 system can control the normal growth development and butenyl-spinosyn biosynthesis of S. pogona, laying an important foundation for revealing the butenyl-spinosyn biosynthetic regulatory mechanism. The limitation of this study is that we have not definitively identified the target genes regulated by RegX3, although several differentially expressed proteins were found based on proteomic data. In addition, the relationship among phosphate concentration, SenX3/RegX3 system, and butenyl-spinosyn biosynthesis needs to be analyzed in depth. Additional experiments such as ChIP-seq and EMSAs are needed to further reveal the biological function of SenX3/RegX3 system.

Resource Availability

Lead Contact

Further information and requests for resources should be directed to and will be fulfilled by the Lead Contact, Liqiu Xia (xialq@hunnu.edu.cn).

Materials Availability

This study did not generate new materials.

Data and Code Availability

In the Supplemental Information, we have provided all the required data needed for reproducibility of this work.

Methods

All methods can be found in the accompanying Transparent Methods supplemental file. The strains, plasmids, and primers used in this study are listed in Tables S1 and S2, respectively.