Multi-omics illuminates the functional significance of previously unknown species in a full-scale landfill leachate treatment plant.

Landfill leachate treatment plants (LLTPs) harbor a vast reservoir of uncultured microbes, yet limited studies have systematically unraveled their functional potentials within LLTPs. Combining 36 metagenomic and 18 metatranscriptomic datasets from a full-scale LLTP, we unveiled a double-edged sword role of unknown species in leachate biotreatment and environmental implication. We identified 655 species-level genome bins (SGBs) spanning 47 bacterial and 3 archaeal phyla, with 75.9 % unassigned to any known species. Over 90 % of up-regulated functional genes in biotreatment units, compared to the leachate influent, were carried by unknown species and actively participated in carbon, nitrogen, and sulfur cycles. Approximately 79 % of the 37,366 carbohydrate active enzymes (CAZymes), with ∼90 % novelty and high expression, were encoded by unknown species, exhibiting great potential in biodegrading carbohydrate compounds linked to human meat-rich diets. Unknown species offered a valuable genetic resource of thousands of versatile, abundant, and actively expressed metabolic gene clusters (MGCs) and biosynthetic gene clusters (BGCs) for enhancing leachate treatment. However, unknown species may contribute to the emission of hazardous N2O/H2S and represented significant reservoirs for antibiotic-resistant pathogens that posed environmental safety risks. This study highlighted the significance of considering both positive and adverse effects of LLTP microbes to optimize LLTP performance.

Prodrug-conjugated tumor-seeking commensals for targeted cancer therapy.

Prodrugs have been explored as an alternative to conventional chemotherapy; however, their target specificity remains limited. The tumor microenvironment harbors a range of microorganisms that potentially serve as tumor-targeting vectors for delivering prodrugs. In this study, we harness bacteria-cancer interactions native to the tumor microbiome to achieve high target specificity for prodrug delivery. We identify an oral commensal strain of Lactobacillus plantarum with an intrinsic cancer-binding mechanism and engineer the strain to enable the surface loading of anticancer prodrugs, with nasopharyngeal carcinoma (NPC) as a model cancer. The engineered commensals show specific binding to NPC via OppA-mediated recognition of surface heparan sulfate, and the loaded prodrugs are activated by tumor-associated biosignals to release SN-38, a chemotherapy compound, near NPC. In vitro experiments demonstrate that the prodrug-loaded microbes significantly increase the potency of SN-38 against NPC cell lines, up to 10-fold. In a mouse xenograft model, intravenous injection of the engineered L. plantarum leads to bacterial colonization in NPC tumors and a 67% inhibition in tumor growth, enhancing the efficacy of SN-38 by 54%.

FELICX: A robust nucleic acid detection method using flap endonuclease and CRISPR-Cas12.

Nucleic acid detection is crucial for monitoring diseases for which rapid, sensitive, and easy-to-deploy diagnostic tools are needed. CRISPR-based technologies can potentially fulfill this need for nucleic acid detection. However, their widespread use has been restricted by the requirement of a protospacer adjacent motif in the target and extensive guide RNA optimization. In this study, we developed FELICX, a technique that can overcome these limitations and provide a useful alternative to existing technologies. FELICX comprises flap endonuclease, Taq ligase and CRISPR-Cas for diagnostics (X) and can be used for detecting nucleic acids and single-nucleotide polymorphisms. This method can be deployed as a point-of-care test, as only two temperatures are needed without thermocycling for its functionality, with the result generated on lateral flow strips. As a proof-of-concept, we showed that up to 0.6 copies/µL of DNA and RNA could be detected by FELICX in 60 min and 90 min, respectively, using simulated samples. Additionally, FELICX could be used to probe any base pair, unlike other CRISPR-based technologies. Finally, we demonstrated the versatility of FELICX by employing it for virus detection in infected human cells, the identification of antibiotic-resistant bacteria, and cancer diagnostics using simulated samples. Based on its unique advantages, we envision the use of FELICX as a next-generation CRISPR-based technology in nucleic acid diagnostics.

Effects of Low Temperature, Nitrogen Starvation and Their Combination on the Photosynthesis and Metabolites of Thermosynechococcus E542: A Comparison Study.

Both low temperature and nitrogen starvation caused chlorosis of cyanobacteria. Here, in this study, for the first time, we compared the effects of low temperature, nitrogen starvation, and their combination on the photosynthesis and metabolites of a thermophilic cyanobacterium strain, Thermosynechococcus E542. Under various culture conditions, the growth rates, pigment contents, and chlorophyll fluorescence were monitored, and the composition of alkanes, lipidomes, and carbohydrates were determined. It was found that low temperature (35 °C) significantly suppressed the growth of Thermosynechococcus E542. Nitrogen starvation at 45 °C and 55 °C did not affect the growth; however, combined treatment of low temperature and nitrogen starvation led to the lowest growth rate and biomass productivity. Both low temperature and nitrogen starvation caused significantly declined contents of pigments, but they resulted in a different effect on the OJIP curves, and their combination led to the lowest pigment contents. The composition of fatty acids and alkanes was altered upon low-temperature cultivation, while nitrogen starvation caused reduced contents of all lipids. The low temperature did not affect carbohydrate contents, while nitrogen starvation greatly enhanced carbohydrate content, and their combination did not enhance carbohydrate content, but led to reduced productivity. These results revealed the influence of low temperature, nitrogen starvation, and their combined treatment for the accumulation of phycobiliproteins, lipids, and carbohydrates of a thermophilic cyanobacterium strain, Thermosynechococcus E542.

Accelerated CO2 Hydration with Thermostable Sulfurihydrogenibium azorense Carbonic Anhydrase-Chitin Binding Domain Fusion Protein Immobilised on Chitin Support.

Carbonic anhydrases (CAs) represent a group of enzymes that catalyse important reactions of carbon dioxide hydration and dehydration, a reaction crucial to many biological processes and environmental biotechnology. In this study we successfully constructed a thermostable fusion enzyme composed of the Sulfurihydrogenibium azorense carbonic anhydrase (Saz_CA), the fastest CA discovered to date, and the chitin binding domain (ChBD) of chitinase from Bacillus circulans. Introduction of ChBD to the Saz_CA had no major impact on the effect of ions or inhibitors on the enzymatic activity. The fusion protein exhibited no negative effects up to 60 °C, whilst the fusion partner appears to protect the enzyme from negative effects of magnesium. The prepared biocatalyst appears to be thermally activated at 60 °C and could be partially purified with heat treatment. Immobilisation attempts on different kinds of chitin-based support results have shown that the fusion enzyme preferentially binds to a cheap, untreated chitin with a large crystallinity index over more processed forms of chitin. It suggests significant potential economic benefits for large-scale deployment of immobilised CA technologies such as CO2 utilisation or mineralisation.

Thermosynechococcus as a thermophilic photosynthetic microbial cell factory for CO2 utilisation.

Thermophilic unicellular cyanobacterium Thermosynechococcus elongatus PKUAC-SCTE542, has been developed as a thermophilic photosynthetic microbial cell factory for CO2 utilisation. The strain exhibits its highest growth rate around 55 °C, can withstand up to 15% CO2, and up to 0.5 M concentration of sodium bicarbonate. The strain is also capable of resisting a 200 ppm concentration of NO and SO2 in simulated flue gasses, and these compounds have a positive effect on its growth. Whole genome sequencing of the strain revealed the presence of numerous forms of active transport of nutrients and additional chaperones acting as the predominant mechanism of strain adaptation to high temperatures. Based on the sequenced genome, two neutral gene insertion sites have been identified and engineered using modular vectors. Site-specific knock-ins and knock-outs have been performed using the spectinomycin resistance gene and proved functional, enabling future application of the strain to produce biofuels and biochemicals from waste CO2.

Complete Genome Sequence and Comparative Analysis of Synechococcus sp. CS-601 (SynAce01), a Cold-Adapted Cyanobacterium from an Oligotrophic Antarctic Habitat.

Marine picocyanobacteria belonging to Synechococcus are major contributors to the global carbon cycle, however the genomic information of its cold-adapted members has been lacking to date. To fill this void the genome of a cold-adapted planktonic cyanobacterium Synechococcus sp. CS-601 (SynAce01) has been sequenced. The genome of the strain contains a single chromosome of approximately 2.75 MBp and GC content of 63.92%. Gene prediction yielded 2984 protein coding sequences and 44 tRNA genes. The genome contained evidence of horizontal gene transfer events during its evolution. CS-601 appears as a transport generalist with some specific adaptation to an oligotrophic marine environment. It has a broad repertoire of transporters of both inorganic and organic nutrients to survive in inhospitable environments. The cold adaptation of the strain exhibited characteristics of a psychrotroph rather than psychrophile. Its salt adaptation strategy is likely to rely on the uptake and synthesis of osmolytes, like glycerol or glycine betaine. Overall, the genome reveals two distinct patterns of adaptation to the inhospitable environment of Antarctica. Adaptation to an oligotrophic marine environment is likely due to an abundance of genes, probably acquired horizontally, that are associated with increased transport of nutrients, osmolytes, and light harvesting. On the other hand, adaptations to low temperatures are likely due to prolonged evolutionary changes.

Temperature-controlled thermophilic bacterial communities in hot springs of western Sichuan, China.

BACKGROUND: Ganzi Prefecture in Western China is situated geographically at the transition regions between Tibetan Plateau and Sichuan Basin in a highly tectonically active boundary area between the India and Eurasia plates. The region hosts various hot springs that span a wide range of temperature from 30 to 98 °C and are located at high altitude (up to 4200 m above sea level) in the region of large geothermal anomalies and active Xianshuihe slip-fault that has been active since Holocene. The site represents a biodiversity reservoir for thermophiles, yet their diversity and relationship to geochemical parameters are largely unknown. In the present work, bacterial diversity and community structure in 14 hot springs of Ganzi were investigated using Illumina MiSeq sequencing. RESULTS: Bacterial community compositions were evidently distinct among the 14 hot springs, and the bacterial communities in hot springs were majorly abundant in phyla Aquificae, Cyanobacteria and Proteobacteria. Both clustering and PCoA analysis suggested the existence of four bacterial community patterns in these hot springs. Temperature contributed to shaping bacterial community structure of hot springs as revealed by correlation analysis. Abundant unassigned-genus sequences detected in this study strongly implied the presence of novel genera or genetic resources in these hot springs. CONCLUSION: The diversity of hot springs of Ganzi prefecture in Western Sichuan, China is evidently shaped by temperature. Interestingly disproportionally abundant unassigned-genus sequences detected in this study show indicate potential of novel genera or phylotypes. We hypothesize that frequent earthquakes and rapidly changing environment might have contributed to evolution of these potentially new lineages. Overall, this study provided first insight into the bacterial diversity of hot springs located in Western Sichuan, China and its comparison with other similar communities worldwide.

Transcriptomic Profiling and Gene Disruption Revealed that Two Genes Related to PUFAs/DHA Biosynthesis May be Essential for Cell Growth of Aurantiochytrium sp.

Aurantiochytrium sp. PKU#SW7 is a thraustochytrid strain that was found to exhibit high potential for docosahexaenoic acid (DHA, C22:6n-3) production. In this work, the transcriptome of Aurantiochytrium sp. PKU#SW7 was analyzed for the study of genes involved in basic metabolic functions and especially in the mechanisms of DHA biosynthesis. Sequence annotation and functional analysis revealed that the strain contains components of fatty acid synthesis (FAS) and polyketide synthase (PKS) pathways. Fatty acid desaturases and elongases were identified as components of FAS pathway, whilst key components of PKS pathway were also found in the cDNA library. The relative contribution of the two pathways to the synthesis of DHA was unknown, as both pathways appeared to be lacking full complement of genes for standalone synthesis of DHA. Further analysis of two putative genes encoding the very-long-chain (3R)-3-hydroxyacyl-CoA dehydratase and dehydrase/isomerase involved in FAS and PKS pathways, respectively, revealed that under various salinity conditions, their relative expression levels changed corresponding to the variation of DHA content in Aurantiochytrium sp. Independent knock outs of these genes in Aurantiochytrium sp. resulted in poor cell growth, probably due to little or no intracellular DHA accumulation. Hence, it can be speculated that both genes are engaged in DHA biosynthesis and DHA in Aurantiochytrium sp. could be produced by jointed actions of both FAS and PKS systems.

Textile Dye Decolorizing Synechococcus PCC7942 Engineered With CotA Laccase.

Cyanobacteria are prokaryotic phototrophs capable of achieving high cellular densities with minimal inputs. These prokaryotic organisms can grow using sunlight as energy source and carbon dioxide as carbon source what makes them promising candidates as microbial cell factories for the production of numerous compounds such as chemicals, fuels, or biocatalysts. In this study, we have successfully designed and constructed using synthetic biology approach two recombinant strains of Synechococcus elongatus PCC7942 for heterologous expression of the industrially relevant Bacillus subtilis CotA laccase. One of the strains (PCC7942-NSI-CotA) was constructed through integration of the laccase gene into neutral site I of the cyanobacterial genome whilst the other (PCC7942-NSII-CotA) targeted neutral site II of the genome. Of the two strains the one with CotA laccase integrated in neutral site II (PCC7942-NSII-CotA) was superior in terms of growth rate and enzymatic activity toward typical laccase substrates: ABTS [2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonate)] and syringaldazine. That may suggest that two of the traditionally used neutral sites of S. elongatus PCC7942 are not equally suitable for the expression of certain transgenes. The PCC7942-NSII-CotA produced protein was capable of decolourising three classes of dyes namely: anthraquinonic-, azo-, and indigoid-type over 7 days of incubation making the strain a potentially useful microbial cell factory for the production of broad-spectrum biodegradation agent. Interestingly, presence of additional synthetic redox mediator ABTS had no effect on the degradation of these dyes.

Abundance and molecular diversity of thraustochytrids in coastal waters of southern China.

Thraustochytrids are unicellular fungi-like (heterotrophic) marine protists that have long been considered to play an important role in the biogeochemical cycles of the coastal oceans. However, the significance of their ecological functions and their diversity in marine ecosystems remain largely unknown. In this report, we examined the spatial and temporal variations of planktonic thraustochytrids, their relationship with other environmental factors, and their diversity in the subtropical coastal waters of China. The abundance of planktonic thraustochytrids ranged from 2.56 × 105 to 17.57 × 105 cells L-1 with highest abundance detected in polluted coastal water in the Spring (March) season. The thraustochytrid biomass was greater than the bacterial biomass in most seawater samples, ranging from 32.29 to 359.51% that of bacterioplankton. The abundance of thraustochytrids appeared to be largely related to that of bacterioplankton and to chemical oxygen demand in water columns. High-throughput sequencing analyses revealed a total of 105 OTUs (97% similarity), which were members of genera Thraustochytrium, Aplanochytrium, Oblongichytrium, Ulkenia, Labyrinthula and undescribed novel phylotypes. Results of this study indicated unprecedented high diversity of labyrinthulomycetes as well as the presence of novel labyrinthulomycete and thraustochytrid lineages, and also provided new information on the significant role of thraustochytrids in microbial food webs in a coastal marine ecosystem.

Astaxanthin-Producing Green Microalga Haematococcus pluvialis: From Single Cell to High Value Commercial Products.

Many species of microalgae have been used as source of nutrient rich food, feed, and health promoting compounds. Among the commercially important microalgae, Haematococcus pluvialis is the richest source of natural astaxanthin which is considered as "super anti-oxidant." Natural astaxanthin produced by H. pluvialis has significantly greater antioxidant capacity than the synthetic one. Astaxanthin has important applications in the nutraceuticals, cosmetics, food, and aquaculture industries. It is now evident that, astaxanthin can significantly reduce free radicals and oxidative stress and help human body maintain a healthy state. With extraordinary potency and increase in demand, astaxanthin is one of the high-value microalgal products of the future.This comprehensive review summarizes the most important aspects of the biology, biochemical composition, biosynthesis, and astaxanthin accumulation in the cells of H. pluvialis and its wide range of applications for humans and animals. In this paper, important and recent developments ranging from cultivation, harvest and postharvest bio-processing technologies to metabolic control and genetic engineering are reviewed in detail, focusing on biomass and astaxanthin production from this biotechnologically important microalga. Simultaneously, critical bottlenecks and major challenges in commercial scale production; current and prospective global market of H. pluvialis derived astaxanthin are also presented in a critical manner. A new biorefinery concept for H. pluvialis has been also suggested to guide toward economically sustainable approach for microalgae cultivation and processing. This report could serve as a useful guide to present current status of knowledge in the field and highlight key areas for future development of H. pluvialis astaxanthin technology and its large scale commercial implementation.