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C-glycosylated flavones (CGFs) are promising candidates as anti-nociceptive compounds. The leaves of bamboo and related crops in the grass family are a largely unexploited bioresource with a wide array of CGFs. We report here pathway-specific enzymes including C-glycosyltransferases (CGTs) and P450 hydroxylases from cereal crops and bamboo species accumulating abundant CGFs. Mining of CGTs and engineering of P450s that decorate the flavonoid skeleton allowed the production of desired CGFs (with yield of 20–40 mg/L) in an Escherichia coli cell factory. We further explored the antinociceptive activity of major CGFs in mice models and identified isoorientin as the most potent, with both neuroanalgesic and anti-inflammatory effects superior to clinical drugs such as rotundine and aspirin. Our discovery of the pain-alleviating flavonoids elicited from bamboo and crop leaves establishes this previously underutilized source, and sheds light on the pathway and pharmacological mechanisms of the compounds., Yuwei Sun, Zhuo Chen, Jingya Yang et al. identify bamboo as a rich source of C-glycosylated flavonoids that reduces pain and inflammation. They identify isoorientin as the most potent C-glycosylated flavonoid, superior to aspirin, and report new enzymes that synthesize pain-alleviating C-glycosylated flavonoids.

It has been suggested that isoprene synthesis by isoprene synthase (IspS) proceeds via a substrate-assisted mechanism. The authors observed a non-enzymatic isoprene formation by Mn2+, which represents the basis of IspS enzyme reaction. Because IspS and many other terpene synthases require Mn2+ metal ions as cofactor, this study characterized the formation reaction for the first time. Metal ions including Mn2+ non-enzymatically produced both isoprene and 2-methyl-3-buten-2-ol (2-MBO) from dimethylallyl pyrophosphate (DMADP). Isoprene formation was most enhanced by Fe2+ and, to a lesser extent, by Mn2+ or Cu2+. Ni2+, Co2+, Mg2+, and Ba2+ exhibited a low activity to generate both isoprene and 2-MBO. The proportion of isoprene and 2-MBO varied with the Mn2+ concentration: isoprene predominated over 2-MBO at a higher Mn2+ concentration. Similarly, isoprene formation by Mn2+ increased exponentially as temperature increased with predominance of isoprene over 2-MBO at higher temperature. Both isoprene and 2-MBO formation was enhanced by acidic and neutral pH compared to alkaline conditions. Molecular dynamic simulation of DMADP suggested that the formation reaction is initiated by deprotonation of hydrogen on allyl terminal carbon by phosphate oxygen and generates carbocation and allyl anion intermediates. This is followed by quenching to produce isoprene or by hydroxyl addition to form 2-MBO. Thus, this study provided an insight into reaction mechanism of isoprene and 2-MBO biosynthesis and highlighted some parts of isoprene emission from terrestrial plants, which could be formed by non-enzymatic mechanism.

Production of the anticancer drug Taxol and its precursors in heterologous hosts is more sustainable than extraction from tissues of yew trees or chemical synthesis. Although attempts to engineer the Taxol pathway in microbes have made significant progress, challenges such as functional expression of plant P450 enzymes remain to be addressed. Here, we introduce taxadiene synthase, taxadiene-5α-hydroxylase, and cytochrome P450 reductase in a high biomass plant Nicotiana benthamiana. Using a chloroplastic compartmentalized metabolic engineering strategy, combined with enhancement of isoprenoid precursors, we show that the engineered plants can produce taxadiene and taxadiene-5α-ol, the committed taxol intermediates, at 56.6 μg g−1 FW and 1.3 μg g−1 FW, respectively. In addition to the tools and strategies reported here, this study highlights the potential of Nicotiana spp. as an alternative platform for Taxol production., Engineering Taxol pathway in microbes needs to overcome the difficulty of expressing plant P450 enzymes. Here, the authors use a compartmentalized metabolic engineering strategy to construct the taxanes production pathway in chloroplasts of Nicotiana benthamiana and realize the production of taxadience-5α-ol.

Aromatic compounds are ubiquitous in nature; they are the building blocks of abundant lignin, and constitute a substantial proportion of synthetic chemicals and organic pollutants. Uptake and degradation of aromatic compounds by bacteria have relevance in bioremediation, bio-based plastic recycling, and microbial conversion of lignocellulosic biomass into high-value commodity chemicals. While remarkable progress has been achieved in understanding aromatic metabolism in biodegraders, the membrane transporter systems responsible for uptake and efflux of aromatic compounds and their degradation products are still poorly understood. Membrane transporters are responsible for the initial recognition, uptake, and efflux of aromatic compounds; thus, in addition to controlling influx and efflux, the transporter system also forms part of stress tolerance mechanisms through excreting toxic metabolites. This review discusses significant advancements in our understanding of the nature and identity of the bacterial membrane transporter systems for aromatics, the molecular and structural basis of substrate recognition, mechanisms of translocation, functional regulation, and biotechnological applications. Most of these developments were enabled through the availability of crystal structures, advancements in computational biophysics, genome sequencing, omics studies, bioinformatics, and synthetic biology. We provide a comprehensive overview of recently reported knowledge on aromatic transporter systems in bacteria, point gaps in our understanding of the underlying translocation mechanisms, highlight existing limitations in harnessing transporter systems in synthetic biology applications, and suggest future research directions.

The diterpenoid paclitaxel (Taxol®) is a blockbuster anticancer agent that was originally isolated from the Pacific yew (Taxus brevifolia) five decades ago. Despite the wealth of information gained over the years on Taxol research, there still remains supply issues to meet increasing clinical demand. Although alternative Taxol production methods have been developed, they still face several drawbacks that cause supply shortages and high production costs. It is highly desired to develop biotechnological production platforms for Taxol, however, there are still gaps in our understanding of the biosynthetic pathway, catalytic enzymes, regulatory and control mechanisms that hamper production of this critical drug by synthetic biology approaches. Over the past 5 years, significant advances were made in metabolic engineering and optimization of the Taxol pathway in different hosts, leading to accumulation of taxane intermediates. Computational and experimental approaches were leveraged to gain mechanistic insights into the catalytic cycle of pathway enzymes and guide rational protein engineering efforts to improve catalytic fitness and substrate/product specificity, especially of the cytochrome P450s (CYP450s). Notable breakthroughs were also realized in engineering the pathway in plant hosts that are more promising in addressing the challenging CYP450 chemistry. Here, we review these recent advances and in addition, we summarize recent transcriptomic data sets of Taxus species and elicited culture cells, and give a bird’s-eye view of the information that can be gleaned from these publicly available resources. Recent mining of transcriptome data sets led to discovery of two putative pathway enzymes, provided many lead candidates for the missing steps and provided new insights on the regulatory mechanisms governing Taxol biosynthesis. All these inferences are relevant to future biotechnological production of Taxol.

Isoprene emissions from six tropical trees were evaluated by “Ping-Pong” method that iteratively optimized the temperature (CT) and light (CL) parameters of the Guenther model, G-93. Six tropical trees; Bauhinia variegate (camel's foot tree), Calophyllum inophyllum (ball tree), Garcinia subelliptica (fukugi tree), Syzygium cumini (black plum), Syzygium samarangense (wax jambu), and Mangifera indica (mango; red and yellow fruit) were exposed to LED light in a phytotron. Saplings of Populus nigra were similarly treated under the same experimental conditions for reference. The irradiance protocol consisted of 26 steps and two cycles of increase and decrease in light intensity. Plants were exposed to a wide variation in light intensity (200–1500 μmol/m2/s), which resulted in leaf temperature variation of 29–40 °C. Predictions of isoprene emission by the optimized G-93 well captured the light and temperature dependent increase and decrease of isoprene emission rate in all tropical trees assessed. Comparatively, G-93 with default parameters showed lower efficacy in predicting emission rates during the ascending phase in the initial round, and at the peak phase in the second round of the irradiation scheme. G-93 with default parameters overestimated emissions at the initial ascending phase and underestimated at the second peak phase. Calculated Q10 values for isoprene emission from tropical trees were much higher than that of the temperate plant P. nigra under same observational conditions. These results strongly imply that isoprene emission behavior of tropical plants deviate from that of temperate plant.

Tropical trees account for most emissions of isoprene, a reactive biogenic volatile organic compound into the atmosphere. The Guenther 1993 (G-93) model is the most widely used algorithm for predicting isoprene emissions by leaves of terrestrial plants. Several studies have reported on the poor performance of the G-93 model in predicting emissions from tropical tree species. To improve the performance of the G-93 model in tropical regions, we carried out diurnal leaf-scale observations of tropical trees Casuarina equisetifolia and Ficus septica outdoors. We developed an iterative method that uses mutual and repetitive step-by-step optimization of the G-93 parameters for temperature (CT) and light (CL) variables using best fit practices, named “Ping-Pong” optimization. Using temperate tree species (Poplar) for comparison, we show that emissions from C. equisetifolia and F. septica had a diversion from predictions of the G-93 formula, especially at high temperatures and high light intensities during mid-day, whilst emissions from temperate tree species were fairly captured by G-93. Results demonstrate that our optimized formulas greatly improved capturing of high light and temperature responses of emission profiles from tropical trees whilst it also performed well for poplar species. Parameterization of the G-93 formula greatly improved its performance on predicting diurnal isoprene emission from tropical trees C. equisetifolia and F. septica; explaining 81–96% of variation up from 73 to 77% explained by default G-93. We propose that there is a need to optimize the G-93 model to more accurately predict regional emissions from tropical ecosystems.

Isoprene is the most abundant type of nonmethane, biogenic volatile organic compound in the atmosphere, and it is produced mainly by terrestrial plants. The tropical tree species Ficus septica Burm. F. (Rosales: Moraceae) has been shown to cease isoprene emissions when exposed to temperatures of 12 °C or lower and to re-induce isoprene synthesis upon subsequent exposure to temperatures of 30 °C or higher for 24 h. To elucidate the regulation of genes underlying the disabling and then induction of isoprene emission during acclimatization to ambient temperature, we conducted gene expression analyses of F. septica plants under changing temperature using quantitative real-time polymerase chain reaction and western blotting. Transcription levels were analyzed for 17 genes that are involved in metabolic pathways potentially associated with isoprene biosynthesis, including isoprene synthase (ispS). The protein levels of ispS were also measured. Changes in transcription and protein levels of the ispS gene, but not in the other assessed genes, showed identical temporal patterns to isoprene emission capacity under the changing temperature regime. The ispS protein levels strongly and positively correlated with isoprene emission capacity (R(2) = 0.92). These results suggest that transcriptional regulation of ispS gave rise to the temporal variation in isoprene emission capacity in response to changing temperature.