Crosstalk of TetR-like regulator SACE_4839 and a nitrogen regulator for erythromycin biosynthesis.

TetR family transcriptional regulators (TFRs) are widespread in actinomycetes, which exhibit diverse regulatory modes in antibiotic biosynthesis. Nitrogen regulators play vital roles in modulation of primary and secondary metabolism. However, crosstalk between TFR and nitrogen regulator has rarely been reported in actinomycetes. Herein, we demonstrated that a novel TFR, SACE_4839, was negatively correlated with erythromycin yield in Saccharopolyspora erythraea A226. SACE_4839 indirectly suppressed erythromycin synthetic gene eryAI and resistance gene ermE and directly inhibited its adjacent gene SACE_4838 encoding a homologue of nitrogen metabolite repression (NMR) regulator NmrA (herein named NmrR). The SACE_4839-binding sites within SACE_4839-nmrR intergenic region were identified. NmrR positively controlled erythromycin biosynthesis by indirectly stimulating eryAI and ermE and directly repressing SACE_4839. NmrR was found to affect growth viability under the nitrogen source supply. Furthermore, NmrR directly repressed glutamine and glutamate utilization-related genes SACE_1623, SACE_5070 and SACE_5979 but activated nitrate utilization-associated genes SACE_1163, SACE_4070 and SACE_4912 as well as nitrite utilization-associated genes SACE_1476 and SACE_4514. This is the first reported NmrA homolog for modulating antibiotic biosynthesis and nitrogen metabolism in actinomycetes. Moreover, combinatorial engineering of SACE_4839 and nmrR in the high-yield S. erythraea WB resulted in a 68.8% increase in erythromycin A production. This investigation deepens the understanding of complicated regulatory network for erythromycin biosynthesis. KEY POINTS: • SACE_4839 and NmrR had opposite contributions to erythromycin biosynthesis. • NmrR was first identified as a homolog of another nitrogen regulator NmrA. • Cross regulation between SACE_4839 and NmrR was revealed.

Uncovering and Engineering a Mini-Regulatory Network of the TetR-Family Regulator SACE_0303 for Yield Improvement of Erythromycin in Saccharopolyspora erythraea.

Erythromycins produced by Saccharopolyspora erythraea have broad-spectrum antibacterial activities. Recently, several TetR-family transcriptional regulators (TFRs) were identified to control erythromycin production by multiplex control modes; however, their regulatory network remains poorly understood. In this study, we report a novel TFR, SACE_0303, positively correlated with erythromycin production in Sac. erythraea. It directly represses its adjacent gene SACE_0304 encoding a MarR-family regulator and indirectly stimulates the erythromycin biosynthetic gene eryAI and resistance gene ermE. SACE_0304 negatively regulates erythromycin biosynthesis by directly inhibiting SACE_0303 as well as eryAI and indirectly repressing ermE. Then, the SACE_0303 binding site within the SACE_0303-SACE_0304 intergenic region was defined. Through genome scanning combined with in vivo and in vitro experiments, three additional SACE_0303 target genes (SACE_2467 encoding cation-transporting ATPase, SACE_3156 encoding a large transcriptional regulator, SACE_5222 encoding α-ketoglutarate permease) were identified and proved to negatively affect erythromycin production. Finally, by coupling CRISPRi-based repression of those three targets with SACE_0304 deletion and SACE_0303 overexpression, we performed stepwise engineering of the SACE_0303-mediated mini-regulatory network in a high-yield strain, resulting in enhanced erythromycin production by 67%. In conclusion, the present study uncovered the regulatory network of a novel TFR for control of erythromycin production and provides a multiplex tactic to facilitate the engineering of industrial actinomycetes for yield improvement of antibiotics.

Polyketide Starter and Extender Units Serve as Regulatory Ligands to Coordinate the Biosynthesis of Antibiotics in Actinomycetes.

Polyketides are one of the largest categories of secondary metabolites, and their biosynthesis is initiated by polyketide synthases (PKSs) using coenzyme A esters of short fatty acids (acyl-CoAs) as starter and extender units. In this study, we discover a universal regulatory mechanism in which the starter and extender units, beyond direct precursors of polyketides, function as ligands to coordinate the biosynthesis of antibiotics in actinomycetes. A novel acyl-CoA responsive TetR-like regulator (AcrT) is identified in an erythromycin-producing strain of Saccharopolyspora erythraea. AcrT shows the highest binding affinity to the promoter of the PKS-encoding gene eryAI in the DNA affinity capture assay (DACA) and directly represses the biosynthesis of erythromycin. Propionyl-CoA (P-CoA) and methylmalonyl-CoA (MM-CoA) as the starter and extender units for erythromycin biosynthesis can serve as the ligands to release AcrT from PeryAI, resulting in an improved erythromycin yield. Intriguingly, anabolic pathways of the two acyl-CoAs are also suppressed by AcrT through inhibition of the transcription of acetyl-CoA (A-CoA) and P-CoA carboxylase genes and stimulation of the transcription of citrate synthase genes, which is beneficial to bacterial growth. As P-CoA and MM-CoA accumulate, they act as ligands in turn to release AcrT from those targets, resulting in a redistribution of more A-CoA to P-CoA and MM-CoA against citrate. Furthermore, based on analyses of AcrT homologs in Streptomyces avermitilis and Streptomyces coelicolor, it is believed that polyketide starter and extender units have a prevalent, crucial role as ligands in modulating antibiotic biosynthesis in actinomycetes. IMPORTANCE Numerous antibiotics are derived from polyketides, whose biosynthesis is accurately controlled by transcriptional regulators that respond to diverse physiological or environmental signals. It is generally accepted that antibiotics or biosynthetic intermediates serve as effectors to modulate their production in actinomycetes. Our study unprecedentedly demonstrates that the direct precursors of polyketide, propionyl-CoA and methylmalonyl-CoA, play a role as ligands to modulate erythromycin biosynthesis in Saccharopolyspora erythraea. More importantly, the two acyl-CoAs as ligands could adjust their own supplies by regulating the acetyl-CoA metabolic pathway so as to well settle the relationship between cellular growth and secondary metabolism. Significantly, polyketide starter and extender units have a universal role as ligands to coordinate antibiotic biosynthesis in actinomycetes. These findings not only expand the understanding of ligand-mediated regulation for antibiotic biosynthesis but also provide new insights into the physiological functions of polyketide starter and extender units in actinomycetes.

[Ligands of TetR family transcriptional regulators: a review].

TetR family transcriptional regulators (TFRs) are widely distributed in bacteria and archaea, and the first discovered TFR was confirmed to control the expression of tetracycline efflux pump in Escherichia coli. TFRs can bind DNAs and ligands. Small molecule ligands can induce conformational changes of TFRs, inhibiting or promoting TFRs to control target gene expression. Currently, TFRs have a wide variety of ligands, including carbohydrates, proteins, fatty acids and their derivatives, metal ions, and so on. Due to the diversity of ligands, TFRs regulate a wide range of physiological processes, from basic carbon metabolism and nitrogen metabolism to quorum sensing and antibiotic biosynthesis. On the basis of the recent studies in our laboratory and the literature, we review here the regulatory mechanism mediated by ligands of TFRs in primary and secondary metabolism, as well as the application of ligands for TFRs in the development of gene route and the activation of antibiotic biosynthesis.

Extensible automated dispersive liquid-liquid microextraction.

In this study, a convenient and extensible automated ionic liquid-based in situ dispersive liquid-liquid microextraction (automated IL-based in situ DLLME) was developed. 1-Octyl-3-methylimidazolium bis[(trifluoromethane)sulfonyl]imide ([C8MIM]NTf2) is formed through the reaction between [C8MIM]Cl and lithium bis[(trifluoromethane)sulfonyl]imide (LiNTf2) to extract the analytes. Using a fully automatic SPE workstation, special SPE columns packed with nonwoven polypropylene (NWPP) fiber, and a modified operation program, the procedures of the IL-based in situ DLLME, including the collection of a water sample, injection of an ion exchange solvent, phase separation of the emulsified solution, elution of the retained extraction phase, and collection of the eluent into vials, can be performed automatically. The developed approach, coupled with high-performance liquid chromatography-diode array detection (HPLC-DAD), was successfully applied to the detection and concentration determination of benzoylurea (BU) insecticides in water samples. Parameters affecting the extraction performance were investigated and optimized. Under the optimized conditions, the proposed method achieved extraction recoveries of 80% to 89% for water samples. The limits of detection (LODs) of the method were in the range of 0.16-0.45 ng mL(-1). The intra-column and inter-column relative standard deviations (RSDs) were <8.6%. Good linearity (r>0.9986) was obtained over the calibration range from 2 to 500 ng mL(-1). The proposed method opens a new avenue for automated DLLME that not only greatly expands the range of viable extractants, especially functional ILs but also enhances its application for various detection methods. Furthermore, multiple samples can be processed simultaneously, which accelerates the sample preparation and allows the examination of a large number of samples.