Functional Characterization and Catalytic Activity Improvement of Borneol Acetyltransferase from Wurfbainia longiligularis.

In plant secondary metabolite biosynthesis, acylation is a diverse physiological process, with BAHD acyltransferases playing an essential role. Borneol acetyltransferase (BAT) is an alcohol acetyltransferase, which catalyzes borneol and acetyl-CoA to synthesize bornyl acetate (BA). However, the enzymes involved in the biosynthesis of BA have so far only been characterized in Wurfbainia villosa, the studies on the WvBATs have only been conducted in vitro, and the catalytic activity was relatively low. In this research, three genes (WlBAT1, WlBAT2, and WlBAT3) have been identified to encode BATs that are capable of acetylating borneol to synthesize BA in vitro. We also determined that WlBAT1 has the highest catalytic efficiency for borneol-type substrates, including (+)-borneol, (-)-borneol, and isoborneol. Furthermore, we found that BATs could catalyze a wide range of substrate types in vitro, but in vivo, they exclusively catalyzed borneol-type substrates. Through molecular simulations and site-directed mutagenesis, it was revealed that residues D32, N36, H168, N297, N355, and H384 are crucial for the catalytic activity of WlBAT1, while the R382I-D385R double mutant of WlBAT1 exhibited an increasing acylation efficiency for borneol-type substrates in vitro and in vivo. These findings offer key genetic elements for the metabolic engineering of plants and synthetic biology to produce BA.

Functional characterization of geranyl/farnesyl diphosphate synthase in Wurfbainia villosa and Wurfbainia longiligularis.

Wurfbainia villosa and Wurfbainia longiligularis are the two primary plant sources of Fructus Amomi, a traditional Chinese medicine. Both plants are rich in volatile terpenoids, including monoterpenes and sesquiterpenes, which are the primary medicinal components of Fructus Amomi. The trans-isopentenyl diphosphate synthase (TIDS) gene family plays a key part in determining terpenoid diversity and accumulation. However, the TIDS gene family have not been identified in W. villosa and W. longiligularis. This study identified thirteen TIDS genes in W. villosa and eleven TIDS genes in W. longiligularis, which may have expanded through segmental replication events. Based on phylogenetic analysis and expression levels, eight candidate WvTIDSs and five WlTIDSs were selected for cloning. Functional characterization in vitro demonstrated that four homologous geranyl diphosphate synthases (GPPSs) (WvGPPS1, WvGPPS2, WlGPPS1, WlGPPS2) and two geranylgeranyl diphosphate synthases (GGPPSs) (WvGGPPS and WlGGPPS) were responsible for catalyzing the biosynthesis of geranyl diphosphate (GPP), whereas two farnesyl diphosphate synthases (FPPSs) (WvFPPS and WlFPPS) catalysed the biosynthesis of the farnesyl diphosphate (FPP). A comparison of six proteins with identified GPPS functions showed that WvGGPPS and WlGGPPS exhibited the highest activity levels. These findings indicate that homologous GPPS and GGPPS together promote the biosynthesis of GPP in W. villosa and W. longiligularis, thus providing sufficient precursors for the synthesis of monoterpenes and providing key genetic elements for Fructus Amomi variety improvement and molecular breeding.

Bioinspired carbon nanotube-based nanofluidic ionic transistor with ultrahigh switching capabilities for logic circuits.

The voltage-gated ion channels, also known as ionic transistors, play substantial roles in biological systems and ion-ion selective separation. However, implementing the ultrafast switchable capabilities and polarity switching of ionic transistors remains a challenge. Here, we report a nanofluidic ionic transistor based on carbon nanotubes, which exhibits an on/off ratio of 104 at operational gate voltage as low as 1 V. By controlling the morphology of carbon nanotubes, both unipolar and ambipolar ionic transistors are realized, and their on/off ratio can be further improved by introducing an Al2O3 dielectric layer. Meanwhile, this ionic transistor enables the polarity switching between p-type and n-type by controlled surface properties of carbon nanotubes. The implementation of constructing ionic circuits based on ionic transistors is demonstrated, which enables the creation of NOT, NAND, and NOR logic gates. The ionic transistors are expected to have profound implications for low-energy consumption computing devices and brain-machine interfacing.

Nudix hydrolase WvNUDX24 is involved in borneol biosynthesis in Wurfbainia villosa.

Borneol, camphor, and bornyl acetate are highly promising monoterpenoids widely used in medicine, flavor, food, and chemical applications. Bornyl diphosphate (BPP) serves as a common precursor for the biosynthesis of these monoterpenoids. Although bornyl diphosphate synthase (BPPS) that catalyzes the cyclization of geranyl diphosphate (GPP) to BPP has been identified in multiple plants, the enzyme responsible for the hydrolysis of BPP to produce borneol has not been reported. Here, we conducted in vitro and in vivo functional characterization to identify the Nudix hydrolase WvNUDX24 from W. villosa, which specifically catalyzes the hydrolysis of BPP to generate bornyl phosphate (BP), and then BP forms borneol under the action of phosphatase. Subcellular localization experiments indicated that the hydrolysis of BPP likely occurs in the cytoplasm. Furthermore, site-directed mutagenesis experiments revealed that four critical residues (R84, S96, P98, and G99) for the hydrolysis activity of WvNUDX24. Additionally, the functional identification of phosphatidic acid phosphatase (PAP) demonstrated that WvPAP5 and WvPAP10 were able to hydrolyze geranylgeranyl diphosphate (GGPP) and farnesyl diphosphate (FPP) to generate geranylgeranyl phosphate (GGP) and farnesyl phosphate (FP), respectively, but could not hydrolyze BPP, GPP, and neryl diphosphate (NPP) to produce corresponding monophosphate products. These findings highlight the essential role of WvNUDX24 in the first step of BPP hydrolysis to produce borneol and provide genetic elements for the production of BPP-related terpenoids through plant metabolic engineering and synthetic biology.

Bio-inspired Two-dimensional Nanofluidic Ionic Transistor for Neuromorphic Signal Processing.

Voltage-gated ion channels prevalent in neurons play important roles in generating action potential and information transmission by responding to transmembrane potential. Fabricating bio-inspired ionic transistors with ions as charge carriers will be crucial for realizing neuro-inspired devices and brain-liking computing. Here, we reported a two-dimensional nanofluidic ionic transistor based on a MXene membrane with sub-1 nm interlayer channels. By applying a gating voltage on the MXene nanofluidic, a transmembrane potential will be generated to active the ionic transistor, which is similar to the transmembrane potential of neuron cells and can be effectively regulated by changing membrane parameters, e.g., thickness, composition, and interlayer spacing. For the symmetric MXene nanofluidic, a high on/off ratio of ~2000 can be achieved by forming an ionic depletion or accumulation zone, contingent on the sign of the gating potential. An asymmetric PET/MXene-composited nanofluidic transitioned the ionic transistor from ambipolar to unipolar, resulting in a more sensitive gate voltage characteristic with a low subthreshold swing of 560 mV/decade. Furthermore, ionic logic gate circuits, including the "NOT", "NAND", and "NOR" gate, were implemented for neuromorphic signal processing successfully, which provides a promising pathway towards highly parallel, low energy consumption, and ion-based brain-like computing.

Ionic Transistors.

Biological voltage-gated ion channels, which behave as life's transistors, regulate ion transport precisely and selectively through atomic-scale selectivity filters to sustain important life activities. By this inspiration, voltage-adaptable ionic transistors that use ions as signal carriers may provide an alternative information processing unit beyond solid-state electronic devices. This review provides a comprehensive overview of the first generation of biomimetic ionic transistors, including their operating mechanisms, device architecture development, and property characterizations. Despite its infancy, significant progress has been made in the applications of ionic transistors in fields such as DNA detection, drug delivery, and ionic circuits. Challenges and prospects of full exploitation of ionic transistors for a broad spectrum of practical applications are also discussed.

Genome-wide identification and functional characterization of borneol dehydrogenases in Wurfbainia villosa.

MAIN CONCLUSION: Genome-wide screening of short-chain dehydrogenases/reductases (SDR) family reveals functional diversification of borneol dehydrogenase (BDH) in Wurfbainia villosa. Wurfbainia villosa is an important medicinal plant, the fruits of which accumulate abundant terpenoids, especially bornane-type including borneol and camphor. The borneol dehydrogenase (BDH) responsible for the conversion of borneol to camphor in W. villosa remains unknown. BDH is one member of short-chain dehydrogenases/reductases (SDR) family. Here, a total of 115 classical WvSDR genes were identified through genome-wide screening. These WvSDRs were unevenly distributed on different chromosomes. Seven candidate WvBDHs based on phylogenetic analysis and expression levels were selected for cloning. Of them, four BDHs can catalyze different configurations of borneol and other monoterpene alcohol substrates to generate the corresponding oxidized products. WvBDH1 and WvBDH2, preferred (+)-borneol to (-)-borneol, producing the predominant ( +)-camphor. WvBDH3 yielded approximate equivalent amount of (+)-camphor and (-)-camphor, in contrast, WvBDH4 generated exclusively (+)-camphor. The metabolic profiles of the seeds showed that the borneol and camphor present were in the dextrorotatory configuration. Enzyme kinetics and expression pattern in different tissues suggested WvBDH2 might be involved in the biosynthesis of camphor in W. villosa. All results will increase the understanding of functional diversity of BDHs.

Comparing genomes of Fructus Amomi-producing species reveals genetic basis of volatile terpenoid divergence.

Wurfbainia longiligularis and Wurfbainia villosa are both rich in volatile terpenoids and are 2 primary plant sources of Fructus Amomi used for curing gastrointestinal diseases. Metabolomic profiling has demonstrated that bornyl diphosphate (BPP)-related terpenoids are more abundant in the W. villosa seeds and have a wider tissue distribution in W. longiligularis. To explore the genetic mechanisms underlying the volatile terpenoid divergence, a high-quality chromosome-level genome of W. longiligularis (2.29 Gb, contig N50 of 80.39 Mb) was assembled. Functional characterization of 17 terpene synthases (WlTPSs) revealed that WlBPPS, along with WlTPS 24/26/28 with bornyl diphosphate synthase (BPPS) activity, contributes to the wider tissue distribution of BPP-related terpenoids in W. longiligularis compared to W. villosa. Furthermore, transgenic Nicotiana tabacum showed that the GCN4-motif element positively regulates seed expression of WvBPPS and thus promotes the enrichment of BPP-related terpenoids in W. villosa seeds. Systematic identification and analysis of candidate TPS in 29 monocot plants from 16 families indicated that substantial expansion of TPS-a and TPS-b subfamily genes in Zingiberaceae may have driven increased diversity and production of volatile terpenoids. Evolutionary analysis and functional identification of BPPS genes showed that BPP-related terpenoids may be distributed only in the Zingiberaceae of monocot plants. This research provides valuable genomic resources for breeding and improving Fructus Amomi with medicinal and edible value and sheds light on the evolution of terpenoid biosynthesis in Zingiberaceae.