Exercise offers short-term and long-term health benefits, including an increased metabolic rate and energy expenditure in myocardium. The newly-discovered exercise-induced myokine, irisin, stimulates conversion of white into brown adipocytes as well as increased mitochondrial biogenesis and energy expenditure. Remarkably, irisin is highly expressed in myocardium, but its physiological effects in the heart are unknown. The objective of this work is to investigate irisin’s potential multifaceted effects on cardiomyoblasts and myocardium. For this purpose, H9C2 cells were treated with recombinant irisin produced in yeast cells (r-irisin) and in HEK293 cells (hr-irisin) for examining its effects on cell proliferation by MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and on gene transcription profiles by qRT-PCR. R-irisin and hr-irisin both inhibited cell proliferation and activated genes related to cardiomyocyte metabolic function and differentiation, including myocardin, follistatin, smooth muscle actin, and nuclear respiratory factor-1. Signal transduction pathways affected by r-irisin in H9C2 cells and C57BL/6 mice were examined by detecting phosphorylation of PI3K-AKT, p38, ERK or STAT3. We also measured intracellular Ca2+ signaling and mitochondrial thermogenesis and energy expenditure in r-irisin-treated H9C2 cells. The results showed that r-irisin, in a certain concentration rage, could activate PI3K-AKT and intracellular Ca2+ signaling and increase cellular oxygen consumption in H9C2 cells. Our study also suggests the existence of irisin-specific receptor on the membrane of H9C2 cells. In conclusion, irisin in a certain concentration rage increased myocardial cell metabolism, inhibited cell proliferation and promoted cell differentiation. These effects might be mediated through PI3K-AKT and Ca2+ signaling, which are known to activate expression of exercise-related genes such as follistatin and myocardin. This work supports the value of exercise, which promotes irisin release.

Pathogenic bacteria demonstrate incredible abilities to evade conventional antibiotics through the development of resistance and formation of dormant, surface-attached biofilms. Therefore, agents that target and eradicate planktonic and biofilm bacteria are of significant interest. We explored a new series of halogenated phenazines (HP) through the use of N-aryl-2nitrosoaniline synthetic intermediates that enabled functionalization of the 3-position of this scaffold. Several HPs demonstrated potent antibacterial and biofilm-killing activities (e.g., HP 29, against methicillin-resistant Staphylococcus aureus: MIC = 0.075 μM; MBEC = 2.35 μM), and transcriptional analysis revealed that HPs 3, 28, and 29 induce rapid iron starvation in MRSA biofilms. Several HPs demonstrated excellent activities against Mycobacterium tuberculosis (HP 34, MIC = 0.80 μM against CDC1551). This work established new SAR insights, and HP 29 demonstrated efficacy in dorsal wound infection models in mice. Encouraged by these findings, we believe that HPs could lead to significant advances in the treatment of challenging infections.

Thaxtomins are a group of phytotoxic diketopiperazines produced by tens of plant pathogenic Streptomyces strains and have received considerable attention as bioherbicide. To synthesize thaxtomin analogue libraries for herbicide development, we here develop an in vitro biocombinatorial approach. We first prepared two recombinant singlemodule multifunctional nonribosomal peptide synthetases (NRPSs), TxtA and TxtB. Biochemical studies revealed that TxtA and TxtB tolerated small substituents on the aromatic moiety of L-Phe and L-Trp, respectively. Intriguingly, TxtB showed a control of the substrate scopes of TxtA and a previously characterized, pathway-specific nitration promoting P450 TxtE. We further demonstrated that the methyltransferase (MT) domain of TxtA and TxtB posed a minimal influence on the assembly of the diketopiperazine core by other domains of these two enzymes, providing a way for structural diversification. The pathway-specific bifunctional P450 TxtC was then recombinantly produced in Escherichia coli after being fused with the reductase domain of P450BM3. Biochemical and kinetic studies indicated that this self-sufficient biocatalyst promoted two hydroxylation reactions first on the aliphatic C14 and then the aromatic C20 of thaxtomin D to sequentially produce thaxtomin B and A. Using these enzymes, a one-pot biocatalytic reaction was developed to synthesize 124 thaxtomin analogues, whose structures were validated in highresolution MS, tandem MS, and sometimes 1D and 2D NMR analysis. Select thaxtomin analogues showed potent herbicidal activities in radish seedling assay. This work demonstrated the feasibility of biocombinatorial synthesis in the creation of natural product-like libraries and provided useful insights into the model of diketopiperazine structural diversity generation in nature, aiding the development of bioactive peptidic compounds in general.

Thaxtomins are virulence factors of most plant-pathogenic strains. Due to their potent herbicidal activity, attractive environmental compatibility, and inherent biodegradability, thaxtomins are key active ingredients of bioherbicides approved by the U.S. Environmental Protection Agency. However, the low yield of thaxtomins in native producers limits their wide agricultural applications. Here, we describe the high-yield production of thaxtomins in a heterologous host. The thaxtomin gene cluster from 87.22 was cloned and expressed in J1074 after chromosomal integration. The production of thaxtomins and nitrotryptophan analogs was observed using liquid chromatography-mass spectrometry (LC-MS) analysis. When the engineered J1074 was cultured in the minimal medium Thx defined medium supplemented with 1% cellobiose (TDMc), the yield of the most abundant and herbicidal analog, thaxtomin A, was 10 times higher than that in 87.22, and optimization of the medium resulted in the highest yield of thaxtomin analogs at about 222 mg/liter. Further engineering of the thaxtomin biosynthetic gene cluster through gene deletion led to the production of multiple biosynthetic intermediates important to the chemical synthesis of new analogs. Additionally, the versatility of the thaxtomin biosynthetic system in J1074 was capitalized on to produce one unnatural fluorinated analog, 5-fluoro-thaxtomin A (5-F-thaxtomin A), whose structure was elucidated by a combination of MS and one-dimensional (1D) and 2D nuclear magnetic resonance (NMR) analyses. Natural and unnatural thaxtomins demonstrated potent herbicidal activity in radish seedling assays. These results indicated that J1074 has the potential to produce thaxtomins and analogs thereof with high yield, fostering their agricultural applications. Thaxtomins are agriculturally valuable herbicidal natural products, but the productivity of native producers is limiting. Heterologous expression of the thaxtomin gene cluster in J1074 resulted in the highest yield of thaxtomins ever reported, representing a significant leap forward in its wide agricultural use. Furthermore, current synthetic routes to thaxtomins and analogs are lengthy, and two thaxtomin biosynthetic intermediates produced at high yields in this work can provide precursors and building blocks to advanced synthetic routes. Importantly, the production of 5-F-thaxtomin A in engineered J1074 demonstrated a viable alternative to chemical methods in the synthesis of new thaxtomin analogs. Moreover, our work presents an attractive synthetic biology strategy to improve the supply of herbicidal thaxtomins, likely finding general applications in the discovery and production of many other bioactive natural products.

Microbes are essential to the global ecosystem, but undesirable microbial growth causes issues ranging from food spoilage and infectious diseases to harmful cyanobacterial blooms. The use of chemicals to control microbial growth has achieved significant success, while specific roles for a majority of essential genes in growth control remain unexplored. Here, we show the growth inhibition of cyanobacterial species by targeting an essential enzyme for the biosynthesis of branched-chain amino acids. Specifically, we report the biochemical, genetic, and structural characterization of dihydroxyacid dehydratase from the model cyanobacterium Synechocystis sp. PCC 6803 (SnDHAD). Our studies suggest that SnDHAD is an oxygen-stable enzyme containing a [2Fe-2S] cluster. Furthermore, we demonstrate that SnDHAD is selectively inhibited in vitro and in vivo by the natural product aspterric acid, which also inhibits the growth of representative bloom-forming Microcystis and Anabaena strains but has minimal effects on microbial pathogens with [4Fe-4S] containing DHADs. This study suggests DHADs as a promising target for the precise growth control of microbes and highlights the exploration of other untargeted essential genes for microbial management.