The evolution of cutinase Est1 based on the clustering strategy and its application for commercial PET bottles degradation.

The rapid increase in global plastic consumption, especially the worldwide use of polyethylene terephthalate (PET), has caused serious pollution problems. Due to the low recycling rate of PET, a substantial amount of waste accumulates in the environment, which prompts a growing focus on enzymatic degradation for its efficiency and environmentally friendliness. This study systematically designed and modified a cutinase, Est1 from Thermobifida alba AHK119, known for its potential of plastic-degradation at high temperatures. Additionally, the introduction of clustering algorithms provided the ability to understand and modify biomolecules, to accelerate the process of finding the optimal mutations. K-means was further proceeded based on the positive mutations. After comprehensive screening for thermostability and activity mutation sites, the dominant mutation Est1_5M (Est1 with the mutations of N213M, T215P, S115P, Q93A, and L91W) exhibited satisfying degradation ability for commercial PET bottles. The results showed that Est1_5M achieved a degradation rate of 90.84% in 72 h, 65-fold higher than the wild type. This study offers reliable theoretical and practical support for the development of efficient PET-degrading enzymes, providing a reference for plastic pollution management.

Aspergillus niger fermentation residues application to produce biochar for the anode of lithium-ion batteries.

Aspergillus niger is widely applied in the fermentation industry, but produce abundant mycelium residues every year. As a kind of solid waste, mycelium residues seriously affect the environment. How to manage and utilize this solid waste is a problem for the fermentation industry. It was reported that many kinds of biomass could be utilized to produce carbon materials, which would be further used to produce lithium-ion rechargeable batteries (LIBs). Here, porous biochar was prepared from A. niger mycelial residues and further used as an anode for LIBs. Since the A. niger mycelium contains abundant nitrogen (5.29%) from its chitosan-dominated cell wall, and silicon (9.63%) from perlite filter aid, respectively, the biochar presented an excellent cycle stability and rate performance when applied as the anode of LIBs. The conclusion of this research shows the wide application prospect of fungal fermentation residues as carbon precursors in energy storage devices. Meanwhile, this investigation provides an alternative management method for A. niger mycelium residues, with which the mycelium residues could be effectively recycled to avoid resource waste and environmental pollution.

The Arabidopsis Mitochondrial Pseudouridine Synthase Homolog FCS1 Plays Critical Roles in Plant Development.

As the most abundant RNA modification, pseudouridylation has been shown to play critical roles in Escherichia coli, yeast and humans. However, its function in plants is still unclear. Here, we characterized leaf curly and small 1 (FCS1), which encodes a pseudouridine synthase in Arabidopsis. fcs1 mutants exhibited severe defects in plant growth, such as delayed development and reduced fertility, and were significantly smaller than the wild type at different developmental stages. FCS1 protein is localized in the mitochondrion. The absence of FCS1 significantly reduces pseudouridylation of mitochondrial 26S ribosomal RNA (rRNA) at the U1692 site, which sits in the peptidyl transferase center. This affection of mitochondrial 26S rRNA may lead to the disruption of mitochondrial translation in the fcs1-1 mutant, causing high accumulation of transcripts but low production of proteins. Dysfunctional mitochondria with abnormal structures were also observed in the fcs1-1 mutant. Overall, our results suggest that FCS1-mediated pseudouridylation of mitochondrial 26S rRNA is required for mitochondrial translation, which is critical for maintaining mitochondrial function and plant development.

The MIEL1-ABI5/MYB30 regulatory module fine tunes abscisic acid signaling during seed germination.

The transcription factor ABSCISIC ACID INSENSITIVE5 (ABI5) plays a crucial role in abscisic acid (ABA) signaling during seed germination. However, how ABI5 is regulated during this process is poorly understood. Here, we report that the ubiquitin E3 ligase MIEL1 and its target transcription factor MYB30 modulate ABA responses in Arabidopsis thaliana during seed germination and seedling establishment via the precise regulation of ABI5. MIEL1 interacts with and ubiquitinates ABI5 to facilitate its degradation during germination. The transcription factor MYB30, whose turnover is mediated by MIEL1 during seed germination, also interacts with ABI5 to interfere with its transcriptional activity. MYB30 functions downstream of MIEL1 in the ABA response, and both are epistatic to ABI5 in ABA-mediated inhibition of seed germination and postgerminative growth. ABA treatment induces the degradation of MIEL1 and represses the interaction between MIEL1 and ABI5/MYB30, thus releasing both ABI5 and MYB30. Our results demonstrate that MIEL1 directly mediates the proteasomal degradation of ABI5 and inhibits its activity via the release of its target protein MYB30, thus ensuring precise ABA signaling during seed germination and seedling establishment.

One-Step Synthesis of 4-Octyl Itaconate through the Structure Control of Lipase.

4-Octyl itaconate is a novel antiviral and immunoregulatory small molecule showing great potential in the treatment of various autoimmune diseases and viral infections. It is difficult to selectively esterify the C4 carboxyl group of itaconate acid via one-step direct esterification using chemical catalysts, while the two-step route with itaconic anhydride as an intermediate is environmentally unfriendly and costly. This research investigated the one-step and green synthesis of 4-octyl itaconate through the structure control of lipase, obtaining 4-octyl itaconate with over 98% yield and over 99% selectivity. Multiscale molecular dynamics simulations were applied to investigate the reaction mechanism. The cavity pocket of lipases resulted in a 4-octyl itaconate selectivity by affecting distribution of substrates toward the catalytic site. Toluene could enhance monoesterification in the C4 carboxyl group and contribute to a nearly 100% conversion from itaconate acid into 4-octyl itaconate by adjusting the catalytic microenvironment around the lipase, producing a shrinkage effect on the channel.

The conversion of linoleic acid into hydroxytetrahydrofuran-structured bio-lubricant.

In this work, a new structured linoleic-based hydroxytetrahydrofuran (HTHF) ester lubricant with excellent properties was developed. A synthesis route through regioselective enzymatic hydration was established, combining highly selective epoxidation with an intramolecular epoxide ring-opening reaction. The results proved that the enzymatic-chemical method is an alternative strategy for the conversion of linoleic acid into bio-lubricants. LiBr was revealed as an efficient catalyst (yields of 95.8%, and selectivity of 98.5%, respectively) for the intramolecular epoxide ring-opening reaction. The tribological properties test indicated that the HTHF bio-lubricants exhibited better performance than the commercial mineral oils. Physicochemical investigation further indicated that the product has a good thermal stability, with the Tonset around 300 °C. The kinematic viscosity and viscosity index indicated that the product is suitable to be applied for lubrication. In contrast with previous findings, this HTHF-structured bio-lubricant oil exhibited a superior low pour point (-64 °C) and provided great potential to be utilized in extreme cold working environments.

ABI5 modulates seed germination via feedback regulation of the expression of the PYR/PYL/RCAR ABA receptor genes.

As abscisic acid (ABA) receptors, PYR1/PYL/RCAR (PYLs) play important roles in ABA-mediated seed germination, but the regulation of PYLs in this process, especially at the transcriptional level, remains unclear. In this study, we found that expression of 11 of 14 PYLs changes significantly during seed germination and is affected by exogenous ABA. Two PYLs, PYL11 and PYL12, both of which are expressed specifically in mature seeds, positively modulate ABA-mediated seed germination. However, ABI5 was found to modulate the PYL11- and PYL12-mediated ABA response. In the abi5-7 mutant, ABA hypersensitivity caused by PYL11 and PYL12 overexpression was totally or partially blocked. By contrast, ABI5 regulates the expression of PYL11 and PYL12 by directly binding to their promoters. Moreover, the expression of eight other PYLs is also affected during the germination of abi5 mutants. Promoter analysis revealed that an ABI5-binding region is present next to the TATA box or initiator box. Together, our data demonstrate the role of PYL11 and PYL12 in seed germination. In addition, the identification of PYLs as targets of ABI5 reveals a role of ABI5 in the feedback regulation of ABA-mediated seed germination.

Biodegradation of low-density polyethylene by Microbulbifer hydrolyticus IRE-31.

Polyethylene (PE) is one of the most widespread plastic materials. Nevertheless, due to its recalcitrance against biological degradation and the presence of toxic additives, landfilled and carelessly disposed PE products have caused serious pollution in the natural environments. In this work, we aimed to investigate the growth characteristics of Microbulbifer hydrolyticus IRE-31 and its application in the biological degradation of low-density PE. The IRE-31 strain was isolated from marine pulp mill wastes rich in lignin which is a natural complex polymer containing also saturated carbon-carbon bonds like in PE. Following 30 days cultivation of the IRE-31 strain, the biodegradation of linear low-density PE particles was evidenced clearly by morphological changes of the polymer surface monitored by scanning electron microscopy and the formation of additional carbonyl groups in the polymer chains indicated by Fourier transform infrared spectroscopy.

The highly-stable immobilization of enzymes on a waste mycelium carrier.

Mycelium is an abundant waste from the fermentation industry, and the environmental problems associated with its required disposal seriously limited the development of fermentation industry. In China, millions of tons of various kinds of mycelium residues were produced each year. Research into providing added-value to mycelium, while avoiding its disposal, is hence of paramount importance. Mycelium can be used as carrier for enzymes, while the enzyme immobilization moreover improves their stability and lifetime performance. Carrier recycling, the natural degradation and disposal of artificial polymer carriers are critical issues in immobilization. This research investigated its use to manufacture a highly-stable immobilized enzyme. An acid pretreatment was employed to enhance the adsorption ability of mycelium, and its adsorption ability was compared with other carriers. Under the optimal conditions, a core-shell immobilized enzyme with porous structure was obtained. The stability and the recycle results of the evaluation indicated the excellent performance of the immobilized enzyme. The mycelium recycling was also investigated to verify the practicability. All the results indicated that the use of a mycelium-based carrier was a promising strategy for the reutilization of the fermentation waste, and this technique provides an alternative way to reduce the total amount of the waste mycelium. Meanwhile, the stability and reusability performance of the mycelium-based immobilization could also decrease the influence of the disposal of the solid waste from denatured enzymes to the environment.

Immobilized bacteria with pH-response hydrogel for self-healing of concrete.

Concrete is significant for construction. A problem in application is the appearance of cracks that will damage its strength. An autogenous crack-healing mechanism based on bacteria receives increasing attention in recent years. The bacteria are able to form calcium carbonate (CaCO3) precipitations in suitable conditions to protect and reinforce the concrete. However, a large number of spores are crushed in aged specimens, resulting in a loss of viability. A new kind of hydrogel crosslinked by alginate, chitosan and calcium ions was introduced in this study. It was observed that the addition of chitosan improved the swelling properties of calcium alginate. Opposite pH response to calcium alginate was observed when the chitosan content in the solution reached 1.0%. With an addition of 1.0% chitosan in hydrogel beads, 10.28% increase of compressive strength and 13.79% increase of flexural strength to the control were observed. The results reveal self-healing properties of concretes. A healing crack of 4 cm length and 1 mm width was observed when using cement PO325, with the addition of bacterial spores (2.54-3.07 × 105/cm3 concrete) encapsulated by hydrogel containing no chitosan.

Hydrolysis of food waste by hot water extraction and subsequent Rhizopus fermentation to fumaric acid.

Food waste is considered a serious global societal problem. How to degrade of food waste in a green and effective way has been to a hot topic. In this work, a method with hot water extraction pretreatment of food waste was investigated and optimized. Under the optimal conditions, more than half of the solid food waste could be transferred to soluble sugars. Meanwhile, in order to improve the tolerance of Rhizopus arrhizus on the food waste hydrolysate, UV combined with chemical mutagenesis were carried out, and a mutant of Rhizopus RH-7-13-807 was obtained. With the mutant strain, the yield of fumaric acid fermented from food waste increased to 1.8 times compared with the original strain, and 23.94 g/L fumaric acid was obtained from the fermentation. Besides, the COD of food waste was evaluated for the degradation of food waste by the Rhizopus RH-7-13-807. The process would decrease the quantity of food waste to be disposed of, and benefit the environment.

Chemoenzymatic Synthesis of Fragrance Compounds from Stearic Acid.

Fatty acids are versatile precursors for fuels, fine chemicals, polymers, perfumes, etc. The properties and applications of fatty acid derivatives depend on chain length and on functional groups and their positions. To tailor fatty acids for desired properties, an engineered P450 monooxygenase has been employed here for enhanced selective hydroxylation of fatty acids. After oxidation of the hydroxy groups to the corresponding ketones, Baeyer-Villiger oxidation could be applied to introduce an oxygen atom into the hydrocarbon chains to form esters, which were finally hydrolyzed to afford either hydroxylated fatty acids or dicarboxylic fatty acids. Using this strategy, we have demonstrated that the high-value-added flavors exaltolide and silvanone supra can be synthesized from stearic acid through a hydroxylation/carbonylation/esterification/hydrolysis/lactonization reaction sequence with isolated yields of about 36 % (for ω-1 hydroxylated stearic acid; 100, 60, 80, 75 % yields for the individual reactions, respectively) or 24 % (for ω-2 hydroxylated stearic acid). Ultimately, we obtained 7.91 mg of exaltolide and 13.71 mg of silvanone supra from 284.48 mg stearic acid.

Outline of the biosynthesis and regulation of ergosterol in yeast.

Sterols are crucial functional components for eukaryotic cell membrane. Due to versatile activities, sterols show wide applications in food and pharmaceutical industries. Ergosterol not only reflects cell growth but also serves as the precursor for manufacturing steroid drugs. To date, the ergosterol biosynthetic pathway in yeast has been reported, and the industrial production of ergosterol is achieved by yeast fermentation or extraction from fungal mycelia. Here, we summarize its biosynthesis, regulation, transportation, and subcellular location of enzymes in yeast. In particular, we review the regulation of ergosterol biosynthesis at transcriptional, translational and post-translational levels. Furthermore, we advocate metabolic engineering and fermentation strategies for high-level production of ergosterol. This study may provide evaluable insights into metabolic engineering of yeast for scaled-up fermentation production of ergosterol or beyond.

Rational design of substrate binding pockets in polyphosphate kinase for use in cost-effective ATP-dependent cascade reactions.

Adenosine-5'-triphosphate (ATP) is the energy equivalent of the living system. Polyphosphate (polyP) is the ancient energy storage equivalent of organisms. Polyphosphate kinases (PPKs) catalyze the polyP formation or ATP formation, to store energy or to regenerate ATP, respectively. However, most PPKs are active only in the presence of long polyPs, which are more difficult and more expensive to generate than the short polyPs. We investigated the PPK preference towards polyPs by site-directed mutagenesis and computational simulation, to understand the mechanism and further design enzymes for effective ATP regeneration using short polyPs for in vitro cascade reactions, which are highly desired for research and applications. The results suggest that the short polyPs inhibit PPK by blocking the ADP-binding pocket. Structural comparison between PPK (Corynebacterium glutamicum) and PPK (Sinorhizobium meliloti) indicates that three amino acid residues, i.e., lysine, glutamate, and threonine, are involved in the activity towards short polyP by fixing the adenosine group of ADP in between the subunits of the dimer, while the terminal phosphate group of ADP still offers an active site, which presents a binding pocket for ADP. A proposed triple mutant PPK (SMc02148-KET) demonstrates significant activity towards short polyP to form ATP from ADP. The obtained high glutathione titer (38.79 mM) and glucose-6-phosphate titer (87.35 mM) in cascade reactions with ATP regeneration using the triple mutant PPK (SMc02148-KET) reveal that the tailored PPK establishes the effective ATP regeneration system for ATP-dependent reactions.

Studies on absorption mechanism and pharmacokinetic properties of albendazole-bile acid conjugate: In vivo and in vitro.

PURPOSE: To improve the oral bioavailability of albendazole (ABZ), a series of albendazole-bile acid conjugates (ABCs) were synthesized. ABC's transmembrane transport mechanism and in vivo pharmacokinetic properties were preliminarily studied. METHODS: The transmembrane transport mechanism of ABCs was studied using the Caco-2 monolayer cell model and intestinal perfusion model. The concentration of ABCs and ABZ were evaluated using High-Performance Liquid Chromatography (HPLC) and HPLC-Mass Spectrometry (HPLC-MS/MS). RESULTS: Compared to ABZ, better permeability was observed for different types and concentrations of ABCs using the Caco-2 monolayer cell model, with ABC-C8 showing the highest permeability. The transmembrane transport of ABCs was affected by ASBT inhibitors, indicating an ASBT-mediated active transport mechanism. Additionally, introducing cholic acid resulted in ABZ no longer being a substrate for P-gp, MRP2, and BCRP, effectively reversing ABZ efflux. In vivo unidirectional intestinal perfusion results in rats showed that ABCs altered the absorption site of ABZ from the jejunum to the ileum. The absorption efficiency of ABCs in each intestinal segment was higher than that of ABZ, and the transmembrane transport efficiency decreased with increasing concentrations of ASBT inhibitors. This further confirmed the presence of both passive diffusion and ASBT-mediated active transport mechanisms in the transport of ABCs. The solubility of ABCs in gastric juice and pharmacokinetics in rats showed that ABZ-C4 exhibited enhanced solubility. Moreover, ABCs significantly increased oral bioavailability compared to ABZ, with ABC-C4 showing an approximately 31-fold increase in bioavailability. CONCLUSION: The transmembrane transport mechanism of ABCs involves a combination of ASBT-mediated active transport and passive diffusion. Moreover, the incorporation of BAs successfully reverses the efflux of ABZ by efflux proteins. Among the synthesized conjugates, ABC-C4 demonstrated superior dissolution behavior both in vitro and in vivo.

Prodrug-inspired adenosine triphosphate-activatable celastrol-Fe(III) chelate for cancer therapy.

Celastrol (CEL), an active compound isolated from the root of Tripterygium wilfordii, exhibits broad anticancer activities. However, its poor stability, narrow therapeutic window and numerous adverse effects limit its applications in vivo. In this study, an adenosine triphosphate (ATP) activatable CEL-Fe(III) chelate was designed, synthesized, and then encapsulated with a reactive oxygen species (ROS)-responsive polymer to obtain CEL-Fe nanoparticles (CEL-Fe NPs). In normal tissues, CEL-Fe NPs maintain structural stability and exhibit reduced systemic toxicity, while at the tumor site, an ATP-ROS-rich tumor microenvironment, drug release is triggered by ROS, and antitumor potency is restored by competitive binding of ATP. This intelligent CEL delivery system improves the biosafety and bioavailability of CEL for cancer therapy. Such a CEL-metal chelate strategy not only mitigates the challenges associated with CEL but also opens avenues for the generation of CEL derivatives, thereby expanding the therapeutic potential of CEL in clinical settings.

MIW1 participates in ABA signaling through the regulation of MYB30 in Arabidopsis.

Seed germination and seedling establishment are critical biological processes, and their underlying molecular mechanisms have practical implications. The ABA signaling during seed germination and early seedling development is negatively regulated by transcription factor MYB30, but its interaction partners and downstream targets are not fully understood. In this study, we identified MIW1 (MYB30-interacting WD40 protein 1), a WD40 protein that could interact with MYB30 and promote its degradation. In the miw1 mutant, the MYB30 protein became more stable. MIW1 enhanced the ABA-mediated inhibition of postgerminative development. The miw1 mutants became hyposensitive to exogenous ABA, and this effect was suppressed by mutations in MYB30. Furthermore, we found that MYB30 negatively regulated the expression of the ABA receptor genes PYR1/PYL/RCARs. The changes in PYLs expression during early seedling development or under ABA treatment became more pronounced in the myb30 mutant. ChIP-qPCR analyses showed MYB30 could directly bind to the promoters of PYL11 and PYL12. Our study reveals that the WD40 protein MIW1 promotes the expression of PYLs by destabilizing MYB30, thus positively regulating the ABA signaling during postgermination in Arabidopsis.

High-Throughput Screening and Directed Evolution of Methionine Adenosyltransferase from Escherichia coli.

S-adenosyl-L-methionine (SAM) is the active form of methionine, which participates in various metabolic reactions and plays a vital role. It is mainly used as a precursor by three key metabolic pathways: trans-methylation, trans-sulfuration, and trans-aminopropylation. Methionine adenosyltransferase (MAT) is the only enzyme to produce SAM from methionine and ATP. However, there is no efficient and accurate method for high-throughput detection of SAM, which is the major obstacles of directed evolution campaigns for MAT. Herein, we established a colorimetric method for directed evolution of MAT based on detecting SAM by using glycine oxidase and glycine/sarcosine N-methyltransferase enzyme. Screening of MAT libraries revealed variant I303V/Q22R with 2.13-fold improved activity towards SAM in comparison to the wild type. Molecular dynamic simulation indicates that the loops more flexible and more conducive to SAM release.

Controllable Star Cationic Poly(Disulfide)s Achieve Genetically Cascade Catalytic Therapy by Delivering Bifunctional Fusion Plasmids.

The absence of effective delivery vectors and suitable multifunctional plasmids limits cancer gene therapy development. The star cationic poly(disulfide)s with ß-cyclodextrin cores (termed ß-CD-g-PSSn ) for caveolae-mediated endocytosis are designed and prepared via mild and controllable disulfide exchange polymerization for high-efficacy cancer therapy. Then, ß-CD-g-PSSn /pDNA complexes are transported to the Golgi apparatus and endoplasmic reticulum. Disulfides in ß-CD-g-PSSn vectors are degraded by glutathione in tumor cells, which not only promotes intracellular pDNA release but also reduces in vitro and in vivo toxicity. One bifunctional fusion plasmid pCATKR, which expresses catalase (CAT) fused to KillerRed (KR) (CATKR) in the same target cell, is also proposed for genetically cascade catalytic therapy. When compared with pCAT-KR (plasmid expressing CAT and KR separately in the same cell), delivered pCATKR decomposes hydrogen peroxide, alleviates tumor hypoxia more effectively, generates stronger reactive oxygen species (ROS) capabilities under moderate irradiation, and leads to robust antitumor cascade photodynamic effects. These impressive results are attributed to fusion protein design, which shortens the distance between CAT and KR catalytic centers and leads to improved ROS production efficiency. This work provides a promising strategy by delivering a catalytic cascade functional plasmid via a high-performance vector with biodegradable and caveolae-mediated endocytosis characteristics.

Excessive consumption of mucin by over-colonized Akkermansia muciniphila promotes intestinal barrier damage during malignant intestinal environment.

Gut microbiota disorders damage the intestinal barrier, which causes intestinal disease. Thus, we screened the microbiota with significant changes using an in situ malignant colorectal cancer (CRC) model. Among the colonies with increased abundance, Akkermansia muciniphila (A. muciniphila) is known for its characteristic of breaking down mucin, which is an essential component of the intestinal barrier. The role of A. muciniphila remains controversial. To investigate the effect of excess A. muciniphila on the intestinal barrier, we established an over-colonized A. muciniphila mouse model by administering a live bacterial suspension after disrupting the original gut microbiome with antibiotics. The results showed that over-colonization of A. muciniphila decreased intestinal mucin content. The mRNA and protein expression levels of tight junction proteins also decreased significantly in the over-colonized A. muciniphila mouse model. Our findings reveal that excess colonization by A. muciniphila breaks the dynamic balance between mucin secretion and degradation, reduces the thickness of the intestinal mucus layer, and damages the intestinal barrier, which would eventually aggravate the development of colitis and CRC. These results will raise awareness about the safety of A. muciniphila serving as a probiotic.