Specific Clearance of Lipopolysaccharide from Blood Based on Peptide Bottlebrush Polymer for Sepsis Therapy.

Lipopolysaccharide (LPS) is the primary bacterial toxin that is vital to the pathogenesis and progression of sepsis associated with extremely high morbidity and mortality worldwide. However, specific clearance of LPS from circulating blood is highly challenging because of the structural complexity and its variation between/within bacterial species. Herein, a robust strategy based on phage display screening and hemocompatible peptide bottlebrush polymer design for specific clearance of targeted LPS from circulating blood is proposed. Using LPS extracted from Escherichia coli as an example, a novel peptide (HWKAVNWLKPWT) with high affinity (KD < 1.0 nм), specificity, and neutralization activity (95.9 ± 0.1%) against the targeted LPS is discovered via iterative affinity selection coupled with endotoxin detoxification screening. A hemocompatible bottlebrush polymer bearing the short peptide [poly(PEGMEA-co-PEP-1)] exhibits high LPS selectivity to reduce circulating LPS level from 2.63 ± 0.01 to 0.78 ± 0.05 EU mL-1 in sepsis rabbits via extracorporeal hemoperfusion (LPS clearance ratio > 70%), reversing the LPS-induced leukocytopenia and multiple organ damages significantly. This work provides a universal paradigm for developing a highly selective hemoadsorbent library fully covering the LPS family, which is promising to create a new era of precision medicine in sepsis therapy.

Modularly engineering Rhodotorula toruloides for α-terpineol production.

α-Terpineol is a monoterpenoid alcohol that has been widely used in the flavor, fragrance, and pharmaceutical industries because of its sensory and biological properties. However, few studies have focused on the microbial production of α-terpineol. The oleaginous yeast Rhodotorula toruloides is endowed with a natural mevalonate pathway and is a promising host in synthetic biology and biorefinery. The primary objective of this work was to engineer R. toruloides for the direct biosynthesis of α-terpineol. The improvement in monoterpenoid production was achieved through the implementation of modular engineering strategies, which included the enhancement of precursor supply, blocking of downstream pathways, and disruption of competing pathways. The results of these three methods showed varying degrees of favorable outcomes in enhancing α-terpineol production. The engineered strain 5L6HE5, with competitive pathway disruption and increased substrate supply, reached the highest product titer of 1.5 mg/L, indicating that reducing lipid accumulation is an efficient method in R. toruloides engineering for terpenoid synthesis. This study reveals the potential of R. toruloides as a host platform for the synthesis of α-terpineol as well as other monoterpenoid compounds.

Secretory expression of ß-1,3-glucomannanase in the oleaginous yeast Rhodosporidium toruloides for improved lipid extraction.

Lipids produced by oleaginous yeasts are considered as sustainable sources for the production of biofuels and oleochemicals. The red yeast Rhodosporidium toruloides can accumulate lipids to over 70% of its dry cell mass. To facilitate lipid extraction, a recombinant ß-1,3-glucomannanase, MAN5C, has been applied to partially breakdown R. toruloides cell wall. In this study, R. toruloides NP11 was engineered for secretory expression of MAN5C to simplify the lipid extraction process. Specifically, a cassette contained a codon-optimized gene MAN5C was integrated into the genome of R. toruloides by Agrobacterium-mediated transformation. The engineered strain NP11-MAN5C was found with proper expression and secretion of active MAN5C, yet no notable compromise in terms of cell growth and lipid production. When NP11-MAN5C cell cultures were extracted with ethyl acetate without any pretreatment, 20% of total lipids were recovered, 4.3-fold higher than that of the parental strain NP11. When the cells were heat-treated followed by extraction with ethyl acetate in the presence of the culture broth supernatants, up to 93% of total lipids were recovered, confirming beneficial effects of MAN5C produced in situ. This study provides a new strategy to engineer oleaginous yeasts for more viable lipid extraction and down-stream processes.

Genetic manipulation of the interconversion between diacylglycerols and triacylglycerols in Rhodosporidium toruloides.

The basidiomycetous yeast Rhodosporidium toruloides (R. toruloides) is an excellent producer for neutral lipids, including triacylglycerols (TAG). Partially because genetic tools for this yeast were less developed, limited efforts were shown to explore its capacity for the production of higher-value lipids such as diacylglycerols (DAG). Here, four genes linked to the interconversion between DAG and TAG were manipulated to promote the production of DAG and free fatty acids (FFA). Among them, three TAG synthesis-related genes, DGA1, LRO1, and ARE1, were down-regulated successively via the RNA interference technology, and an endogenous TAG lipase encoded by TGL5 was fused with LDP1 and over-expressed to convert TAG into DAG and FFA. Results showed that those engineered R. toruloides strains grew normally under nutrient-rich conditions but notably slower than the parental strain NP11 in the lipid production stage. When cultivated in nitrogen-limited media, engineered strains were able to produce total lipids with improved contents of DAG and FFA by up to two-fold and three-fold, respectively. Further correlation analysis between lipid composition and cell density indicated that the formation of TAG correlated positively with cell growth; however, other lipids including DAG did negatively. This study offered valuable information and strains to engineer R. toruloides for advanced production of fatty acid derivatives.

Engineering the Oleaginous Yeast Rhodosporidium toruloides for Improved Resistance Against Inhibitors in Biomass Hydrolysates.

Conversion of lignocellulosic biomass into lipids and related chemicals has attracted much attention in the past two decades, and the oleaginous yeast Rhodosporidium toruloides has been widely used in this area. While R. toruloides species naturally have physiological advantages in terms of substrate utilization, lipid accumulation, and inhibitor resistance, reduced lipid production and cell growth are noticed when biomass hydrolysates are used as feedstocks. To improve the robustness of R. toruloides, here, we devised engineered strains by overexpressing genes responsible for phenolic compound degradation. Specifically, gene expression cassettes of the manganese peroxidase gene (MNP) and versatile peroxidase gene (VP) were constructed and integrated into the genome of R. toruloides NP11. A series of engineered strains were evaluated for lipid production in the presence of typical phenolic inhibitors. The results showed that R. toruloides strains with proper expression of MNP or VP indeed grew faster in the presence of vanillin and 5-hydroxymethylfurfural than the parental strain. When cultivated in concentrated mode biomass hydrolysates, the strain VP18 had improved performance as the cell mass and lipid content increased by 30% and 25%, respectively. This study provides more robust oleaginous yeast strains for microbial lipid production from lignocellulosic biomass, and similar efforts may be used to devise more advanced lipid producers.

Engineering Rhodosporidium toruloides for limonene production.

BACKGROUND: Limonene is a widely used monoterpene in the production of food, pharmaceuticals, biofuels, etc. The objective of this work was to engineer Rhodosporidium toruloides as a cell factory for the production of limonene. RESULTS: By overexpressing the limonene synthase (LS), neryl pyrophosphate synthase (NPPS)/geranyl pyrophosphate synthase and the native hydroxy-methyl-glutaryl-CoA reductase (HMGR), we established a baseline for limonene production based on the mevalonate route in Rhodosporidium toruloides. To further enhance the limonene titer, the acetoacetyl-CoA thiolase/HMGR (EfMvaE) and mevalonate synthase (EfMvaS) from Enterococcus faecalis, the mevalonate kinase from Methanosarcina mazei (MmMK) and the chimeric enzyme NPPS-LS were introduced in the carotenogenesis-deficient strain. The resulting strains produced a maximum limonene titer of 393.5 mg/L. CONCLUSION: In this study, we successfully engineered the carotenogenesis yeast R. toruloides to produce limonene. This is the first report on engineering R. toruloides toward limonene production based on NPP and the fusion protein SltNPPS-CltLS. The results demonstrated that R. toruloides is viable for limonene production, which would provide insights into microbial production of valuable monoterpenes.

Reduction of lipid-accumulation of oleaginous yeast Rhodosporidium toruloides through CRISPR/Cas9-mediated inactivation of lipid droplet structural proteins.

The basidiomycetous yeast Rhodosporidium toruloides is an important chassis organism for producing microbial lipids and terpenoids. However, excess carbon flux flows towards lipid synthesis than terpenoid synthesis. Thus, it is essential to limit lipid accumulation so that R. toruloides can be explored as an advanced cell factory for producing non-lipid derivatives. In this study, we knocked out two lipid droplet (LD) structural proteins (Ldp1 and Cals) of R. toruloides NP11 through the CRISPR/Cas9 system to reduce lipid production. The results showed that lipid content of LD protein-disrupted strains dropped by over 40%. LDP1-disrupted mutants harbored small-sized LDs. This study provided valuable information to study about microbial lipid metabolism and platform strains for constructing advanced cell factories.

Performance of HABR + MSABP system for the treatment of dairy wastewater and analyses of microbial community structure and low excess sludge production.

The potential of the system, a hybrid anaerobic baffled reactor (HABR) coupled with a multi-stage active biological process (MSABP) reactor, for simulated dairy wastewater at various temperature, hydraulic retention time (HRT), and pH was investigated. Percentage removals of chemical oxygen demand (COD) and NH4+ were optimized using response surface methodology. Under optimized conditions (temperature, 33 °C; HRT, 24 h; pH, 7.35), the removal efficiencies of COD and NH4+ were 99.89% and 97.83%, respectively. Miseq sequencing analysis exhibited that the anaerobic segment of the system was dominated by fermentation and acetogenic bacteria, and in the aerobic segment, microorganisms involved in the nitrogen cycle were significantly enriched. Meanwhile, it could be found that the excess sludge production of the process was much lower than that of other bio-processes. The average excess sludge production rate was 0.025-0.05 g SS/g COD removed under different organic loadings.

Effects of vibration on anammox-enriched biofilm in a high-loaded upflow reactor.

An upflow biofilm reactor was operated for 211 days to investigate the effects of vibration on anammox treatment performance. With vibration, the highest nitrogen removal rates (20 kg-N·m-3·d-1) were obtained on day 180. Since the vibration could directly applied on the biofilm, it could release the dinitrogen gas accumulated in the biofilm timely and reduce the internal mass transfer resistance sharply. The specific anammox activity increased by more than 3 times with a higher vibration intensity. Meanwhile, the unique random motion caused by mechanical vibration promotes the production of extracellular proteins. Moreover, the VSS reached 20.97 g·L-1 which was 1.6 times higher than the control reactor. Such enrichment method resulted in a hard and thick anammox biofilm with a special granular morphology, and the nitrite tolerance concentration could reach 500 mg-N·L-1. Operated with an adequate vibration intensity could maintain the biofilm thickness and conducive to improve the stability of the reactor. In addition, this technique also allowed the microorganisms inside the biofilm and those on the surface to reach the same culture conditions. Base on the batch experiments, intermittent vibration caused a decrease in energy consumption from about 7.757 (kW·h)·(kg-N)-1 in group 0-Lv7(60-60) to 0.912 (kW·h)·(kg-N)-1 in group 0-Lv7(5-60). Compared to the internal recycle without vibration, the energy consumption fell by a slice over 65%. Furthermore, the high-throughput sequencing results showed that the relative abundance of Candidatus Kuenenia in reactor 1 increased from 13.2% to 43.9%.

Performance analysis and optimization of ammonium removal in a new biological folded non-aerated filter reactor.

A new type of biological folded non-aerated filter (BFNAF) was found to be superior and feasible for the treatment of NH4+-N wastewater. It was constructed with the folded structure suitable for the nylon biomass carrier. The advantages of the BFNAF included low energy consumption, long reaction path, large biofilm surface area and non-clogging compared to the traditional biological aerated filter. In this study, the effects of hydraulic retention time (HRT), and the influent NH4+-N concentration on the performance of BFNAF were investigated and optimized by the response surface methodology. Under the optimal operating condition (HRT, 10 h; NH4+-N concentration, 52 mg/L), the removal efficiency and removal rate were 94.62 ±â€¯0.63% and 0.106 kg-NH4+ m-3 day-1, respectively. The results showed that the BFNAF reactor could remove NH4+-N from wastewater and realized the nitrification process effectively under natural ventilation conditions.

Nitrogen removal from wastewater via simultaneous nitrification and denitrification using a biological folded non-aerated filter.

A conventional biological filter has been shown to be a viable method for removing nitrogenous compounds from wastewater, but it still has many disadvantages. In this study, a biological folded non-aerated filter (BFNAF) was designed, and its feasibility for nitrogen-loaded wastewater treatment has been confirmed. Effects of the HRT and the COD/N ratio on the performance of BFNAF were investigated. Through response surface method, when the COD/N ratio and the HRT were 5.39 and 10.83 h, removal efficiencies of NH4+, COD and TN reached maximum values of 88.62 ±â€¯0.81%, 76.12 ±â€¯0.57%, and 50.48 ±â€¯1.02%, respectively. In addition, it was found that several denitrifying bacteria, such as Azoarcus, Arcobacter, Flavobacterium, along with many ammonia-oxidizing bacteria and nitrite-oxidizing bacteria, co-existed in the community of the biofilm. All the results showed that the BFNAF could realize the simultaneous nitrification and denitrification (SND) process effectively.

A novel anammox process combined with vibration technology.

This study investigated a fixed bed anammox bioreactor that uses vibration techniques to treat synthetic inorganic wastewater. Continuous experiments indicated that the activity elevation period could be shorten to one third, when the nitrogen removal rate (NRR) reached 1 kg·N/m3·d with vibration. R2 achieved the maximum NRR of 3.3 kg·N/m3·d under the resonance state, which was 1.8 times higher than the control reactor. Analysis of vibration intensity suggested that anammox activity would be great improved with the increasing vibration. These results indicated that vibration played a key role in system performance. Furthermore, high-throughput sequencing showed that the reactor with the vibration had a higher proportion of anammox bacteria, which increased 7 times than the biofilm formation phase. Meanwhile, the proportion of Proteobacteria and Chloroflexi decreased by 37.1% and 7.78%, respectively. These results suggest that vibration could increase the anammox treatment performance and provide a better condition for the anammox bacteria.

A new interaction between proximal and distal C-terminus of Cav1.2 channels.

Cardiac Cav1.2 channels, coupling membrane stimulation to intracellular Ca2+ signaling, are regulated by multiple cytoplasmic factors, such as calmodulin (CaM), phosphorylation, Ca2+, ATP and intramolecular fragments of the channel. The interaction between distal and proximal C-terminal regulatory domains (DCRD and PCRD) of Cav1.2 channel is suggested to inhibit the channel activity, while PKA-mediated phosphorylation facilitates Cav1.2 channel by releasing such an interaction. Here, we report that the interaction between the distal C-terminus (CT3) and the proximal C-terminus (CT1) are inhibited by CaM in a Ca2+-dependent manner. Furthermore, CT3D (a short CT3 with DCRD truncated) interacts with CT1B (a short CT1 with EF-hand and PCRD truncated), revealing a new interaction between distal and proximal C-terminus. Ca2+/CaM inhibited the binding of CT3D to CT1B more strongly than the binding between CT3 and CT1, implying that the interaction of DCRD/PCRD (in CT3/CT1) might cooperate with the binding of CT3D to CT1B. We name the new CT1B-binding region of CT3D as CaM-competitive domain (CCD). The electrophysiological experiments show that CT3D inhibits while CT1B facilitates Cav1.2 channel activity in inside-out patches in guinea-pig ventricular myocytes. These results suggest that distal C-terminus inhibits Cav1.2 channel through modulation of the CaM-binding property of the channels.