Discovery, Structure, and Engineering of a cis-Geranylfarnesyl Diphosphate Synthase.

cis-Prenyltransferases (cis-PTs) catalyze the sequential head-to-tail condensation of isopentenyl diphosphate (IPP) to allylic diphosphates, producing mixed E-Z prenyl diphosphates of varying lengths; however, the specific enzymes synthesizing cis-C25 prenyl diphosphates have not been identified. Herein, we present the discovery and characterization of a cis-geranylfarnesyl diphosphate synthase (ScGFPPS) from Streptomyces clavuligerus. This enzyme demonstrates high catalytic proficiency in generating six distinct cis-polyisoprenoids, including three C25 and three C20 variants. We determined the crystal structure of ScGFPPS. Additionally, we unveil the crystal structure of nerylneryl diphosphate synthase (NNPS), known for synthesizing an all-cis-C20 polyisoprenoid. Comparative structural analysis of ScGFPPS and NNPS has identified key differences that influence product specificity. Through site-directed mutagenesis, we have identified eight single mutations that significantly refine the selectivity of ScGFPPS for cis-polyisoprenoids. Our findings not only expand the functional spectrum of cis-PTs but also provide a structural comparison strategy in cis-PTs engineering.

Discovery and biosynthesis of bacterial drimane-type sesquiterpenoids from Streptomyces clavuligerus.

Drimane-type sesquiterpenoids (DMTs) are characterized by a distinctive 6/6 bicyclic skeleton comprising the A and B rings. While DMTs are commonly found in fungi and plants, their presence in bacteria has not been reported. Moreover, the biosynthetic pathways for DMTs have been primarily elucidated in fungi, with identified P450s only acting on the B ring. In this study, we isolated and characterized three bacterial DMTs, namely 3ß-hydroxydrimenol (2), 2α-hydroxydrimenol (3), and 3-ketodrimenol (4), from Streptomyces clavuligerus. Through genome mining and heterologous expression, we identified a cav biosynthetic gene cluster responsible for the biosynthesis of DMTs 2-4, along with a P450, CavA, responsible for introducing the C-2 and C-3 hydroxy groups. Furthermore, the substrate scope of CavA revealed its ability to hydroxylate drimenol analogs. This discovery not only broadens the known chemical diversity of DMTs from bacteria, but also provides new insights into DMT biosynthesis in bacteria.

Contracting pore channels of a magnesium-based metal-organic framework by decorating methyl groups for effective Xe/Kr separation.

A new magnesium-based metal-organic framework with unprecedented short-chain secondary building units and ultra-micropore channels approaching the kinetic diameters of Xe is fabricated by decorating methyl groups on ligands. Due to the contracted pores, this MOF exhibits very high selectivity values for Xe/Kr, which ranks it among the top porous absorbents.

Cytochrome P450-Mediated Skeleton Rearrangement of Taxadiene in an Engineered Escherichia coli System.

In this study, we constructed a taxadiene overproduction platform and identified a cytochrome P450, CYP701A8, that activates the inert C-H bonds in taxadiene to produce three oxidized products (1-3). Compound 1 possesses a newly identified 1 (15→11) abeotaxane skeleton, while 3 features a distinctive 6/10-fused carbocyclic core with an α,ß-unsaturated ketone moiety. Our quantum computations suggested a carbocation-driven rearrangement in the formation of 1. These results support CYP701A8 as a promising biocatalyst for the generation of novel taxane diterpenoids.

High speciation rate of niche specialists in hot springs.

Ecological and evolutionary processes simultaneously regulate microbial diversity, but the evolutionary processes and their driving forces remain largely unexplored. Here we investigated the ecological and evolutionary characteristics of microbiota in hot springs spanning a broad temperature range (54.8-80 °C) by sequencing the 16S rRNA genes. Our results demonstrated that niche specialists and niche generalists are embedded in a complex interaction of ecological and evolutionary dynamics. On the thermal tolerance niche axis, thermal (T) sensitive (at a specific temperature) versus T-resistant (at least in five temperatures) species were characterized by different niche breadth, community abundance and dispersal potential, consequently differing in potential evolutionary trajectory. The niche-specialized T-sensitive species experienced strong temperature barriers, leading to completely species shift and high fitness but low abundant communities at each temperature ("home niche"), and such trade-offs thus reinforced peak performance, as evidenced by high speciation across temperatures and increasing diversification potential with temperature. In contrast, T-resistant species are advantageous of niche expansion but with poor local performance, as shown by wide niche breadth with high extinction, indicating these niche generalists are "jack-of-all-trades, master-of-none". Despite of such differences, the T-sensitive and T-resistant species are evolutionarily interacted. Specifically, the continuous transition from T-sensitive to T-resistant species insured the exclusion probability of T-resistant species at a relatively constant level across temperatures. The co-evolution and co-adaptation of T-sensitive and T-resistant species were in line with the red queen theory. Collectively, our findings demonstrate that high speciation of niche specialists could alleviate the environmental-filtering-induced negative effect on diversity.

Effects of Mineral on Taxonomic and Functional Structures of Microbial Community in Tengchong Hot Springs via in-situ cultivation.

Diverse mineralogical compositions occur in hot spring sediments, but the impact of minerals on the diversity and structure of microbial communities remains poorly elucidated. In this study, different mineral particles with various chemistries (i.e., hematite, biotite, K-feldspar, quartz, muscovite, aragonite, serpentine, olivine, barite, apatite, and pyrite) were incubated for ten days in two Tengchong hot springs, one alkaline (pH ~ 8.34) with a high temperature (~ 82.8 °C) (Gumingquan, short as GMQ) and one acidic (pH ~ 3.63) with a relatively low temperature (~ 43.3 °C) (Wenguangting, short as WGT), to determine the impacts of minerals on the microbial communities taxonomic and functional diversities. Results showed that the mineral-associated bacterial taxa differed from those of the bulk sediment samples in the two hot springs. The relative abundance of Proteobacteria, Euryarchaeota, and Acidobacteria increased in all minerals, indicating that these microorganisms are apt to colonize on solid surfaces. The α-diversity indices of the microbial communities on the mineral surfaces in the WGT were higher than those from the bulk sediment samples (p < 0.05), which may be caused by the stochastically adhering process on the mineral surface during 10-day incubation, different from the microbial community in sediment which has experienced long-term environmental and ecological screening. Chemoheterotrophy increased with minerals incubation, which was high in most cultured minerals (the relative contents were 5.8 - 21.4%). Most notably, the sulfate respiration bacteria (mainly related to Desulfobulbaceae and Syntrophaceae) associated with aragonite in the acidic hot spring significantly differed from other minerals, possibly due to the pH buffering effect of aragonite providing more favorable conditions for their survival and proliferation. By comparison, aragonite cultured in the alkaline hot spring highly enriched denitrifying bacteria and may have promoted the nitrogen cycle within the system. Collectively, we speculated that diverse microbes stochastically adhered on the surface of minerals in the water flows, and the physicochemical properties of minerals drove the enrichment of certain microbial communities and functional groups during the short-term incubation. Taken together, these findings thereby provide novel insights into mechanisms of community assembly and element cycling in the terrestrial hydrothermal system associated with hot springs.

Discovery, Structure, and Mechanism of a Class II Sesquiterpene Cyclase.

Terpene cyclases (TCs), extraordinary enzymes that create the structural diversity seen in terpene natural products, are traditionally divided into two classes, class I and class II. Although the structural and mechanistic features of class I TCs are well-known, the corresponding details in class II counterparts have not been fully characterized. Here, we report the genome mining discovery and structural characterization of two class II sesquiterpene cyclases (STCs) from Streptomyces. These drimenyl diphosphate synthases (DMSs) are the first STCs shown to possess ß,γ-didomain architecture. High-resolution X-ray crystal structures of DMS from Streptomyces showdoensis (SsDMS) in complex with both a farnesyl diphosphate and Mg2+ unveiled an induced-fit mechanism, with an unprecedented Mg2+ binding mode, finally solving one of the lingering questions in class II TC enzymology. This study supports continued genome mining for novel bacterial TCs and provides new mechanistic insights into canonical class II TCs that will lead to advances in TC engineering and synthetic biology.

A critical review of mineral-microbe interaction and co-evolution: mechanisms and applications.

Mineral-microbe interactions play important roles in environmental change, biogeochemical cycling of elements and formation of ore deposits. Minerals provide both beneficial (physical and chemical protection, nutrients, and energy) and detrimental (toxic substances and oxidative pressure) effects to microbes, resulting in mineral-specific microbial colonization. Microbes impact dissolution, transformation and precipitation of minerals through their activity, resulting in either genetically controlled or metabolism-induced biomineralization. Through these interactions, minerals and microbes co-evolve through Earth history. Mineral-microbe interactions typically occur at microscopic scale but the effect is often manifested at global scale. Despite advances achieved through decades of research, major questions remain. Four areas are identified for future research: integrating mineral and microbial ecology, establishing mineral biosignatures, linking laboratory mechanistic investigation to field observation, and manipulating mineral-microbe interactions for the benefit of humankind.

Efficient production of clerodane and ent-kaurane diterpenes through truncated artificial pathways in Escherichia coli.

The clerodane and ent-kaurane diterpenoids are two typical categories of diterpenoid natural products with complicated polycyclic carbon skeletons and significant pharmacological activities. Despite exciting advances in organic chemistry, access to these skeletons is still highly challenging. Using synthetic biology to engineer microbes provides an innovative alternative to bypass synthetic challenges. In this study, we constructed two truncated artificial pathways to efficiently produce terpentetriene and ent-kaurene, two representative clerodane and ent-kaurane diterpenes, in Escherichia coli. Both pathways depend on the exogenous addition of isoprenoid alcohol to reinforce the supply of IPP and DMAPP via two sequential phosphorylation reactions. Optimization of these constructs provided terpentetriene and ent-kaurene titers of 66 ± 4 mg/L and 113 ± 7 mg/L, respectively, in shake-flask fermentation. The truncated pathways to overproduce clerodane and ent-kaurane skeletons outlined here may provide an attractive route to prepare other privileged diterpene scaffolds.

Minerals Determined a Special Ecological Niche and Selectively Enriched Microbial Species from Bulk Water Communities in Hot Springs.

Minerals provide physical niches and supply nutrients or serve as electron donors/acceptors for microorganism survival and growth, and thus minerals and microbes co-evolved. Yet, little is known about how sediment minerals impact microbial community assembly in hot springs and to what extent mineralogical composition influences microbial community composition and diversity. Here the influences of minerals on thermophiles in Tengchong hot springs were revealed by network analysis of field samples, as well as in-situ microcosm experiments with minerals. A molecular ecological network was constructed based on high throughput sequencing data of 16S rRNA gene, with a combination of water geochemistry and sedimentary mineralogical compositions. Six modules were identified and this highly modular network structure represents the microbial preference to different abiotic factors, consequently resulting in niche partitioning in sedimentary communities in hot springs. Diverse mineralogical compositions generated special niches for microbial species. Subsequently, the in-situ microcosm experiments with four minerals (aragonite, albite, K-feldspar, and quartz) and spring water were conducted in a silicate-hosted alkaline spring (i.e., Gmq) and a carbonate-hosted neutral hot spring (i.e., Gxs) for 70 days. Different microbial preferences were observed among different mineral types (carbonate versus silicate). Aragonite microcosms in Gmq spring enriched archaeal genera Sulfophobococcus and Aeropyrum within the order Desulfurococcales by comparison with both in-situ water and silicate microcosms. Sulfophobococcus was also accumulated in Gxs aragonite microcosms, but the contribution to overall dissimilarity is much lower than that in Gmq spring. Besides, Caldimicrobium was a bacterial genus enriched in Gxs aragonite microcosms, in contrast to in-situ water and silicate microcosms, whereas Candidatus Kryptobacter and Thermus were more abundant in silicate microcosms. The differences in microbial accumulations among different mineral types in the same spring implied that mineral chemistry may exert extra deterministic selective pressure in drawing certain species from the bulk water communities, in addition to stochastic absorption on mineral surface. Taken together, our results highlight the special niche partitioning determined by mineralogical compositions and further confirm that minerals could be used as "fishing bait" to enrich certain rare microbial species.

Temperature and microbial interactions drive the deterministic assembly processes in sediments of hot springs.

Terrestrial geothermal ecosystems, as a representative of extreme environments, exhibit a variety of geochemical gradients, and their microbes are thought to be under high stress through environmental selection. However, it is still unclear how stochasticity and biotic interactions contribute to the microbial community assembly in hot springs. Here, we investigated the assembly processes and co-occurrence patterns of microbiota (i.e. bacteria and archaea) in both water and sediments sampled from fifteen hot springs in the Tengchong area, Southwestern of China, using 16S rRNA gene sequencing combined with multivariate ecological and statistical methods. These hot springs harbored more specialists than non-geothermal ecosystems, which are well-adapted to the extreme conditions, as shown by extremely high nearest-taxon index (NTI) and narrower niche width. Habitat differentiation led to the differences in microbial diversity, species-interactions, and community assembly between water and sediment communities. The sediment community showed stronger phylogenetic clustering and was primarily governed by heterogeneous selection, while undominated stochastic processes and dispersal limitation were the major assembly processes in the water community. Temperature and ferrous iron were the major factors mediating the balance of stochastic and deterministic assembly processes in sediment communities, as evidenced by how divergences in temperature and ferrous iron increased the proportion of determinism. Microbial interactions in sediments contributed to deterministic community assembly, as indicated by more complex associations and greater responsiveness to environmental change than water community. These findings uncover the ecological processes underlying microbial communities in hot springs, and provide potential insight into understanding the mechanism to maintain microbial diversity in extreme biospheres.

HRESIMS-guided isolation of aspidosperma-scandine type bisindole alkaloids from Melodinus cochinchinensis and their anti-inflammatory and cytotoxic activities.

The Melodinus species have been proved to be good resources of bisindole alkaloids. Six bisindole alkaloids were isolated from the leaves and stems of Melodinus cochinchinensis (Lour.) Merr. guided by HRESIMS data analysis. Among them, melokhanines K-M, epi-scandomelonine, and epi-scandomeline possessed aspidosperma-scandine skeleton linked by a C-C bond while meloyine II had a scandine-scandine skeleton. The structures were established by extensive spectroscopic analysis of their HRESIMS and NMR data. Melokhanines K-M were undescribed compounds, while epi-scandomelonine, epi-scandomeline and meloyine II were known compounds, which were reported from Melodinus species for the first time. The anti-inflammatory and cytotoxic activities of the isolates were also evaluated in vitro. Melokhanine K and meloyine II showed potent inhibitory activity on the production of nitric oxide, interleukin-6, and tumor necrosis factor-α in LPS-induced RAW 264.7 macrophages, whereas epi-scandomelonine and epi-scandomeline exhibited certain cytotoxic activity against MOLT-4 cells with IC50 values 5.2 and 1.5 µM, respectively.