Isoprene is a valuable precursor for synthetic rubber and a signature product of terpenoid pathways. Here, we developed an isoprene biosensor by employing a TbuT transcriptional regulator of Ralstonia pickettii to express a fluorescent reporter gene in response to intracellular isoprene in engineered Escherichia coli. The TbuT regulator recognizes isoprene as its less-preferred effector molecule; thus, we amplified the reporter gene expression using a T7 RNA polymerase-mediated transcriptional cascade and iteratively tuned the promoter transcribing tbuT to improve the sensitivity for detecting isoprene. When the engineered E. coli cells expressed heterologous genes for isoprene biosynthesis, the intracellular isoprene was expelled and the tbuT transcription factor was subsequently activated, leading to gfp expression. The chromosomal isoprene biosensor showed a linear correlation between GFP fluorescence and intracellular isoprene concentration. Using this chromosomal isoprene biosensor, we successfully identified the highest isoprene producer among four different E. coli strains producing different amounts of isoprene. The isoprene biosensor presented here can enable high-throughput screening of isoprene synthases and metabolic pathways for efficient and sustainable production of bioisoprene in engineered microbes.

Staphylococcus aureus is one of the most important pathogens causing chronic biofilm infections. These are becoming more difficult to treat owing to drug resistance, particularly because S. aureus biofilms limit the efficacy of antimicrobial agents, leading to high morbidity and mortality. In the present study, we screened for inhibitors of S. aureus biofilm formation using a natural product library from the Korea Chemical Bank (KCB). Screening by crystal violet-based biomass staining assay identified hit compounds. Further examination of antibiofilm properties of these compounds was conducted and led to the identification of celastrol and telithromycin. In vitro, both celastrol and telithromycin were toxic to planktonic S. aureus and also active against a clinical methicillin-resistant S. aureus (MRSA) isolate. The effect of the compounds on preformed biofilms of clinical MRSA isolates was evaluated by confocal laser scanning microscopy (CLSM), which revealed the absence of typical biofilm architecture. In addition, celastrol and telithromycin inhibited the production of extracellular protein at selected sub-MIC concentrations, which revealed the reduced extracellular polymeric substance (EPS) secretion. Celastrol exhibited greater cytotoxicity than telithromycin. These data suggest that the hit compounds, especially telithromycin, could be considered novel inhibitors of S. aureus biofilm. Although the mechanisms of the effects on S. aureus biofilms are not fully understood, our data suggest that telithromycin could be a useful adjuvant therapeutic agent for S. aureus biofilm-related infections.

SummaryTargeted gene regulation is indispensable for reprogramming a cellular network to modulate a microbial phenotype. Here, we adopted the type II CRISPR interference (CRISPRi) system for simple and efficient regulation of target genes in Pseudomonas putida KT2440. A single CRISPRi plasmid was generated to express a nuclease‐deficient Cas9 gene and a designed single guide RNA, under control of l‐rhamnose‐inducible Prha BAD and the constitutive Biobrick J23119 promoter respectively. Two target genes were selected to probe the CRISPRi‐mediated gene regulation: exogenous green fluorescent protein on the multicopy plasmid and endogenous glpR on the P. putida KT2440 chromosome, encoding GlpR, a transcriptional regulator that represses expression of the glpFKRD gene cluster for glycerol utilization. The CRISPRi system successfully repressed the two target genes, as evidenced by a reduction in the fluorescence intensity and the lag phase of P. putida KT2440 cell growth on glycerol. Furthermore, CRISPRi‐mediated repression of glpR improved both the cell growth and glycerol utilization, resulting in the enhanced production of mevalonate in an engineered P. putida KT2440 harbouring heterologous genes for the mevalonate pathway. CRISPRi is expected to become a robust tool to reprogram P. putida KT2440 for the development of microbial cell factories producing industrially valuable products.

Bisabolol is a functional ingredient in various health and cosmetic products and has antibacterial, antiinflammatory, and wound healing properties. (−)-α-Bisabolol is chemically synthesized and produced by steam distillation of essential oils extracted from Brazilian Candeia (Eremanthus erythropappus). To sustainably produce pure (−)-α-bisabolol, we previously engineered Escherichia coli to produce 9.1 g/L (−)-α-bisabolol via heterologous mevalonate pathways and (−)-αbisabolol synthase (BOS) from German chamomile, Matricaria recutita (MrBOS). BOS has only been reported in MrBOS and Brazilian Candeia (EeBOS). The limited availability of BOS has made it difficult to achieve high titer and yield and large-scale (−)-α-bisabolol production. We identified a novel BOS in globe artichoke (CcBOS) and examined its functionality in vitro and in vivo. CcBOS showed higher catalytic efficiency and (−)-α-bisabolol production rates than those from MrBOS or EeBOS. In fedbatch fermentation, CcBOS generated the highest reported (−)-α-bisabolol titer to date (23.4 g/L). These results may facilitate economically viable industrial (−)-α-bisabolol production.