AKR7A5 knockout promote acute liver injury by inducing inflammatory response, oxidative stress and apoptosis in mice.

Alcohol liver disease has become a worldwide critical health problem. The ingested alcohol could be converted into acetaldehyde or combined with free fatty acids to induce the endoplasmic reticulum oxidative stress in the liver. Coincidentally, AKR7A5 has both aldehyde detoxification and antioxidant effects. Therefore, we discuss the possible role and mechanism of AKR7A5 in the acute alcohol injury of mice liver. There were four experiment groups, the C57BL/6 mice of wild-type mice (WT) or AKR7A5-/- mice (KO) were intragastrically administrated with saline or 50% ethanol at 14 mL/kg, respectively. Compared to the WT + alcohol group, abnormal liver function, disordered hepatic cord, severe congestion in the hepatic sinus and the space of the hepatic cord, occurrence of oxidative stress, DNA damage and different expressions of apoptosis-related proteins were detected in the KO + alcohol group. Meanwhile, the biological process enrichment analysis showed that the down-regulated proteins were related to the metabolism of fatty acid, the up-regulated proteins positive regulation of reactive oxygen species metabolic process, negative regulation of coagulation and haemostasis. In conclusion, single ethanol binge combined with the absence of AKR7A5 caused more severe inflammatory response, oxidative stress, apoptosis of endogenous pathways, abnormal lipids metabolism and disordered coagulation in mice liver.

Capturing a methanogenic carbon monoxide dehydrogenase/acetyl-CoA synthase complex via cryogenic electron microscopy.

Approximately two-thirds of the estimated one-billion metric tons of methane produced annually by methanogens is derived from the cleavage of acetate. Acetate is broken down by a Ni-Fe-S-containing A-cluster within the enzyme acetyl-CoA synthase (ACS) to carbon monoxide (CO) and a methyl group (CH3+). The methyl group ultimately forms the greenhouse gas methane, whereas CO is converted to the greenhouse gas carbon dioxide (CO2) by a Ni-Fe-S-containing C-cluster within the enzyme carbon monoxide dehydrogenase (CODH). Although structures have been solved of CODH/ACS from acetogens, which use these enzymes to make acetate from CO2, no structure of a CODH/ACS from a methanogen has been reported. In this work, we use cryo-electron microscopy to reveal the structure of a methanogenic CODH and CODH/ACS from Methanosarcina thermophila (MetCODH/ACS). We find that the N-terminal domain of acetogenic ACS, which is missing in all methanogens, is replaced by a domain of CODH. This CODH domain provides a channel for CO to travel between the two catalytic Ni-Fe-S clusters. It generates the binding surface for ACS and creates a remarkably similar CO alcove above the A-cluster using residues from CODH rather than ACS. Comparison of our MetCODH/ACS structure with our MetCODH structure reveals a molecular mechanism to restrict gas flow from the CO channel when ACS departs, preventing CO escape into the cell. Overall, these long-awaited structures of a methanogenic CODH/ACS reveal striking functional similarities to their acetogenic counterparts despite a substantial difference in domain organization.

Resolving the metabolism of monolignols and other lignin-related aromatic compounds in Xanthomonas citri.

Lignin, a major plant cell wall component, has an important role in plant-defense mechanisms against pathogens and is a promising renewable carbon source to produce bio-based chemicals. However, our understanding of microbial metabolism is incomplete regarding certain lignin-related compounds like p-coumaryl and sinapyl alcohols. Here, we reveal peripheral pathways for the catabolism of the three main lignin precursors (p-coumaryl, coniferyl, and sinapyl alcohols) in the plant pathogen Xanthomonas citri. Our study demonstrates all the necessary enzymatic steps for funneling these monolignols into the tricarboxylic acid cycle, concurrently uncovering aryl aldehyde reductases that likely protect the pathogen from aldehydes toxicity. It also shows that lignin-related aromatic compounds activate transcriptional responses related to chemotaxis and flagellar-dependent motility, which might play an important role during plant infection. Together our findings provide foundational knowledge to support biotechnological advances for both plant diseases treatments and conversion of lignin-derived compounds into bio-based chemicals.

Phylogenetic diversity of putative nickel-containing carbon monoxide dehydrogenase-encoding prokaryotes in the human gut microbiome.

Although the production of carbon monoxide (CO) within the human body has been detected, only two CO-utilizing prokaryotes (CO utilizers) have been reported in the human gut. Therefore, the phylogenetic diversity of the human gut CO-utilizing prokaryotes remains unclear. Here, we unveiled more than a thousand representative genomes containing genes for putative nickel-containing CO dehydrogenase (pCODH), an essential enzyme for CO utilization. The taxonomy of genomes encoding pCODH was expanded to include 8 phyla, comprising 82 genera and 248 species. In contrast, putative molybdenum-containing CODH genes were not detected in the human gut microbial genomes. pCODH transcripts were detected in 97.3 % (n=110) of public metatranscriptome datasets derived from healthy human faeces, suggesting the ubiquitous presence of prokaryotes bearing transcriptionally active pCODH genes in the human gut. More than half of the pCODH-encoding genomes contain a set of genes for the autotrophic Wood-Ljungdahl pathway (WLP). However, 79 % of these genomes commonly lack a key gene for the WLP, which encodes the enzyme that synthesizes formate from CO2, suggesting that potential human gut CO-utilizing prokaryotes share a degenerated gene set for WLP. In the other half of the pCODH-encoding genomes, seven genes, including putative genes for flavin adenine dinucleotide-dependent NAD(P) oxidoreductase (FNOR), ABC transporter and Fe-hydrogenase, were found adjacent to the pCODH gene. None of the putative genes associated with CO-oxidizing respiratory machinery, such as energy-converting hydrogenase genes, were found in pCODH-encoding genomes. This suggests that the human gut CO utilization is not for CO removal, but potentially for fixation and/or biosynthesis, consistent with the harmless yet continuous production of CO in the human gut. Our findings reveal the diversity and distribution of prokaryotes with pCODH in the human gut microbiome, suggesting their potential contribution to microbial ecosystems in human gut environments.

Improving the production of 22-hydroxy-23,24-bisnorchol-4-ene-3-one in Mycolicibacterium smegmatis.

The 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) is a C22 steroid synthon of pharmaceutical interest that can be produced as a lateral end-product of the catabolism of natural sterols (e.g., cholesterol or phytosterols). This work studies the role of an aldehyde dehydrogenase coded by the MSMEG_6563 gene of Mycolicibacterium smegmatis, named msRed, in 4-HBC production. This gene is located contiguously to the MSMEG_6561 encoding the aldolase msSal which catalyses the retroaldol elimination of acetyl-CoA of the metabolite intermediate 22-hydroxy-3-oxo-cholest-4-ene-24-carboxyl-CoA to deliver 3-oxo-4-pregnene-20-carboxyl aldehyde (3-OPA). We have demonstrated that msRed reduces 3-OPA to 4-HBC. Moreover, the role of msOpccR reductase encoded by MSMEG_1623 was also explored confirming that it also performs the reduction of 3-OPA into 4-HBC, but less efficiently than msRed. To obtain a M. smegmatis 4-HBC producer strain we deleted MSMEG_5903 (hsd4A) gene in strain MS6039-5941 (ΔkshB1, ΔkstD1) that produces 4-androstene-3,17-dione (AD) from natural sterols (cholesterol or phytosterols). The triple MS6039-5941-5903 mutant was able to produce 9 g/L of 4-HBC from 14 g/L of phytosterols in 2 L bioreactor, showing a productivity of 0.140 g/L h-1. To improve the metabolic flux of sterols towards the production of 4-HBC we have cloned and overexpressed the msSal and msRed enzymes in the MS6039-5941-5903 mutant rendering a production titter of 12.7 g/L with a productivity of 0.185 g/L h-1, and demonstrating that the new recombinant strain has a great potential for its industrial application.

Metabolic engineering of Komagataella phaffii for the efficient utilization of methanol.

BACKGROUND: Komagataella phaffii, a type of methanotrophic yeast, can use methanol, a favorable non-sugar substrate in eco-friendly bio-manufacturing. The dissimilation pathway in K. phaffii leads to the loss of carbon atoms in the form of CO2. However, the ΔFLD strain, engineered to lack formaldehyde dehydrogenase-an essential enzyme in the dissimilation pathway-displayed growth defects when exposed to a methanol-containing medium. RESULTS: Inhibiting the dissimilation pathway triggers an excessive accumulation of formaldehyde and a decline in the intracellular NAD+/NADH ratio. Here, we designed dual-enzyme complex with the alcohol oxidase1/dihydroxyacetone synthase1 (Aox1/Das1), enhancing the regeneration of the formaldehyde receptor xylulose-5-phosphate (Xu5P). This strategy mitigated the harmful effects of formaldehyde accumulation and associated toxicity to cells. Concurrently, we elevated the NAD+/NADH ratio by overexpressing isocitrate dehydrogenase in the TCA cycle, promoting intracellular redox homeostasis. The OD600 of the optimized combination of the above strategies, strain DF02-1, was 4.28 times higher than that of the control strain DF00 (ΔFLD, HIS4+) under 1% methanol. Subsequently, the heterologous expression of methanol oxidase Mox from Hansenula polymorpha in strain DF02-1 resulted in the recombinant strain DF02-4, which displayed a growth at an OD600 4.08 times higher than that the control strain DF00 in medium containing 3% methanol. CONCLUSIONS: The reduction of formaldehyde accumulation, the increase of NAD+/NADH ratio, and the enhancement of methanol oxidation effectively improved the efficient utilization of a high methanol concentration by strain ΔFLD strain lacking formaldehyde dehydrogenase. The modification strategies implemented in this study collectively serve as a foundational framework for advancing the efficient utilization of methanol in K. phaffii.

ALDH1A3-acetaldehyde metabolism potentiates transcriptional heterogeneity in melanoma.

Cancer cellular heterogeneity and therapy resistance arise substantially from metabolic and transcriptional adaptations, but how these are interconnected is poorly understood. Here, we show that, in melanoma, the cancer stem cell marker aldehyde dehydrogenase 1A3 (ALDH1A3) forms an enzymatic partnership with acetyl-coenzyme A (CoA) synthetase 2 (ACSS2) in the nucleus to couple high glucose metabolic flux with acetyl-histone H3 modification of neural crest (NC) lineage and glucose metabolism genes. Importantly, we show that acetaldehyde is a metabolite source for acetyl-histone H3 modification in an ALDH1A3-dependent manner, providing a physiologic function for this highly volatile and toxic metabolite. In a zebrafish melanoma residual disease model, an ALDH1-high subpopulation emerges following BRAF inhibitor treatment, and targeting these with an ALDH1 suicide inhibitor, nifuroxazide, delays or prevents BRAF inhibitor drug-resistant relapse. Our work reveals that the ALDH1A3-ACSS2 couple directly coordinates nuclear acetaldehyde-acetyl-CoA metabolism with specific chromatin-based gene regulation and represents a potential therapeutic vulnerability in melanoma.

Beneficial base substitutions in Escherichia coli fucO gene for enhancement of glycolic acid production.

Microbial production of glycolic acid (GA) from ethylene glycol is extensively used in a variety of industries because ethylene glycol is not only an inexpensive raw material but also the main component of industrial wastes. In this study, we produced GA from ethylene glycol using Escherichia coli overexpressing the endogenous 1,2-propanediol oxidoreductase (fucO) and lactaldehyde dehydrogenase (aldA) genes. To increase GA productivity, we screened a random mutant library generated using an error-prone polymerase chain reaction of fucO and obtained FucO mutants MF2-9 and MF6-9 with enhanced GA production in Lysogeny Broth medium containing 800 mM ethylene glycol. MF2-9 contained three amino acid substitutions (D23E, E222K, and G363S) and two synonymous mutations (coding DNA [c.] 93G > A and c.1131T > C) in fucO. MF6-9 contained one amino acid substitution (L377H) in FucO. An amino acid substitution (L377H) and a single synonymous mutation (c.1131T > C) in fucO contributed to the enhancement in GA production. Notably, cell lysates from E. coli harboring a synonymous mutation (c.1131T > C) or amino acid substitution (L377H) in fucO showed that only AldA activity was 1.3-fold higher than that of the cell lysate from E. coli harboring the wild-type fucO. We confirmed that c.1131T > C and L377H mutations increased aldA expression in E. coli. Analysis of mRNA levels and simulation of mRNA stabilization indicated that base substitutions at positions c.1130T, which corresponds to L377H amino acid substitution, and c.1131T increased aldA expression due to partial destabilization of the mRNA. These findings will be useful for the large-scale microbial production of GA from industrial waste.

FOXO1 induced fatty acid oxidation in hepatic cells by targeting ALDH1L2.

BACKGROUND AND AIM: Lipid metabolism disorder is the primary feature of numerous refractory chronic diseases. Fatty acid oxidation, an essential aerobic biological process, is closely related to the progression of NAFLD. The forkhead transcription factor FOXO1 has been reported to play an important role in lipid metabolism. However, the molecular mechanism through which FOXO1 regulates fatty acid oxidation remains unclear. METHODS: Transcriptomic analysis was performed to examine the cellular expression profile to determine the functional role of FOXO1 in HepG2 cells with palmitic acid (PA)-induced lipid accumulation. FOXO1-binding motifs at the promoter region of aldehyde dehydrogenase 1 family member L2 (ALDH1L2) were predicted via bioinformatic analysis and confirmed via luciferase reporter assay. Overexpression of ALDH1L2 was induced to recover the impaired fatty acid oxidation in FOXO1-knockout cells. RESULTS: Knockout of FOXO1 aggravated lipid deposition in hepatic cells. Transcriptomic profiling revealed that knockout of FOXO1 increased the expression of genes associated with fatty acid synthesis but decreased the expression of carnitine palmitoyltransferase1a (CPT1α) and adipose triglyceride lipase (ATGL), which contribute to fatty acid oxidation. Mechanistically, FOXO1 was identified as a transcription factor of ALDH1L2. Knockout of FOXO1 significantly decreased the protein expression of ALDH1L2 and CPT1α in vitro and in vivo. Furthermore, overexpression of ALDH1L2 restored fatty acid oxidation in FOXO1-knockout cells. CONCLUSION: The findings of this study indicate that FOXO1 modulates fatty acid oxidation by targeting ALDH1L2.

Structural identification and comprehension of human ALDH1L1-Gossypol complex.

The folate metabolism enzyme ALDH1L1 catalyzed 10-formyltetrahydrofolate to tetrahydrofolate and CO2. Non-small cell lung cancer cells (NSCLC) strongly express ALDH1L1. Gossypol binds to an allosteric site and disrupts the folate metabolism by preventing NADP+ binding. The Cryo-EM structures of tetrameric C-terminal aldehyde dehydrogenase human ALDH1L1 complex with gossypol were examined. Gossypol-bound ALDH1L1 interfered with NADP+ by shifting the allosteric site of the structural conformation, producing a closed-form NADP+ binding site. In addition, the inhibition activity of ALDH1L1 was targeted with gossypol in NSCLC. The gossypol treatment had anti-cancer effects on NSCLC by blocking NADPH and ATP production. These findings emphasize the structure characterizing ALDH1L1 with gossypol.

S-nitrosoglutathione reductase alleviates morphine analgesic tolerance by restricting PKCα S-nitrosation.

Morphine, a typical opiate, is widely used for controlling pain but can lead to various side effects with long-term use, including addiction, analgesic tolerance, and hyperalgesia. At present, however, the mechanisms underlying the development of morphine analgesic tolerance are not fully understood. This tolerance is influenced by various opioid receptor and kinase protein modifications, such as phosphorylation and ubiquitination. Here, we established a murine morphine tolerance model to investigate whether and how S-nitrosoglutathione reductase (GSNOR) is involved in morphine tolerance. Repeated administration of morphine resulted in the down-regulation of GSNOR, which increased excessive total protein S-nitrosation in the prefrontal cortex. Knockout or chemical inhibition of GSNOR promoted the development of morphine analgesic tolerance and neuron-specific overexpression of GSNOR alleviated morphine analgesic tolerance. Mechanistically, GSNOR deficiency enhanced S-nitrosation of cellular protein kinase alpha (PKCα) at the Cys78 and Cys132 sites, leading to inhibition of PKCα kinase activity, which ultimately promoted the development of morphine analgesic tolerance. Our study highlighted the significant role of GSNOR as a key regulator of PKCα S-nitrosation and its involvement in morphine analgesic tolerance, thus providing a potential therapeutic target for morphine tolerance.

S-nitrosylation may inhibit the activity of COP1 in plant photomorphogenesis.

Protein S-nitrosylation, which is defined by the covalent attachment of nitric oxide (NO) to the thiol group of cysteine residues, is known to play critical roles in plant development and stress responses. NO promotes seedling photomorphogenesis and NO emission is enhanced by light. However, the function of protein S-nitrosylation in plant photomorphogenesis is largely unknown. E3 ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) and transcription factor ELONGATED HYPOCOTYL 5 (HY5) antagonistically regulate seedling photomorphogenesis. COP1 inhibits plant photomorphogenesis by targeting photomorphogenic promoters like HY5 for 26S proteasome degradation. Here, we report that COP1 is S-nitrosylated in vitro. Mass spectrometry analyses revealed that two evolutionarily well conserved residues, cysteine 425 and cysteine 607, in the WD40 domain of COP1 are S-nitrosylated. S-nitrosylated glutathione (GSNO) is an important physiological NO donor for protein S-nitrosylation. The Arabidopsis (Arabidopsis thaliana) gsnor1-3 mutant, which accumulates higher level of GSNO, accumulated higher HY5 levels than wildtype (WT), indicating that COP1 activity is inhibited. Protein S-nitrosylation can be reversed by Thioredoxin-h5 (TRXh5) in plants. Indeed, COP1 interacts directly with TRXh5 and its close homolog TRXh3. Moreover, catalase 3 (CAT3) acts as a transnitrosylase that transfers NO to its target proteins like GSNO reductase (GSNOR). We found that CAT3 interacts with COP1 in plants. Taken together, our data indicate that the activity of COP1 is likely inhibited by NO via S-nitrosylation to promote the accumulation of HY5 and photomorphogenesis.

Coupling CO2 Reduction and Acetyl-CoA Formation: The Role of a CO Capturing Tunnel in Enzymatic Catalysis.

The bifunctional CO-dehydrogenase/acetyl-CoA synthase (CODH/ACS) complex couples the reduction of CO2 to the condensation of CO with a methyl moiety and CoA to acetyl-CoA. Catalysis occurs at two sites connected by a tunnel transporting the CO. In this study, we investigated how the bifunctional complex and its tunnel support catalysis using the CODH/ACS from Carboxydothermus hydrogenoformans as a model. Although CODH/ACS adapted to form a stable bifunctional complex with a secluded substrate tunnel, catalysis and CO transport is even more efficient when two monofunctional enzymes are coupled. Efficient CO channeling appears to be ensured by hydrophobic binding sites for CO, which act in a bucket-brigade fashion rather than as a simple tube. Tunnel remodeling showed that opening the tunnel increased activity but impaired directed transport of CO. Constricting the tunnel impaired activity and CO transport, suggesting that the tunnel evolved to sequester CO rather than to maximize turnover.

Pharmacologic Targeting of Histone H3K27 Acetylation/BRD4-dependent Induction of ALDH1A3 for Early-phase Drug Tolerance of Gastric Cancer.

Anticancer drug-tolerant persister (DTP) cells at an early phase of chemotherapy reshape refractory tumors. Aldehyde dehydrogenase 1 family member A3 (ALDH1A3) is commonly upregulated by various anticancer drugs in gastric cancer patient-derived cells (PDC) and promotes tumor growth. However, the mechanism underlying the generation of ALDH1A3-positive DTP cells remains elusive. Here, we investigated the mechanism of ALDH1A3 expression and a combination therapy targeting gastric cancer DTP cells. We found that gastric cancer tissues treated with neoadjuvant chemotherapy showed high ALDH1A3 expression. Chromatin immunoprecipitation (ChIP)-PCR and ChIP sequencing analyses revealed that histone H3 lysine 27 acetylation was enriched in the ALDH1A3 promoter in 5-fluorouracil (5-FU)-tolerant persister PDCs. By chemical library screening, we found that the bromodomain and extraterminal (BET) inhibitors OTX015/birabresib and I-BET-762/molibresib suppressed DTP-related ALDH1A3 expression and preferentially inhibited DTP cell growth. In DTP cells, BRD4, but not BRD2/3, was recruited to the ALDH1A3 promoter and BRD4 knockdown decreased drug-induced ALDH1A3 upregulation. Combination therapy with 5-FU and OTX015 significantly suppressed in vivo tumor growth. These observations suggest that BET inhibitors are efficient DTP cell-targeting agents for gastric cancer treatment. SIGNIFICANCE: Drug resistance hampers the cure of patients with cancer. To prevent stable drug resistance, DTP cancer cells are rational therapeutic targets that emerge during the early phase of chemotherapy. This study proposes that the epigenetic regulation by BET inhibitors may be a rational therapeutic strategy to eliminate DTP cells.

CO Oxidation Mechanism of Silver-Substituted Mo/Cu CO-Dehydrogenase - Analogies and Differences to the Native Enzyme.

The aerobic oxidation of carbon monoxide to carbon dioxide is catalysed by the Mo/Cu-containing CO-dehydrogenase enzyme in the soil bacterium Oligotropha carboxidovorans, enabling the organism to grow on the small gas molecule as carbon and energy source. It was shown experimentally that silver can be substituted for copper in the active site of Mo/Cu CODH to yield a functional enzyme. In this study, we employed QM/MM calculations to investigate whether the reaction mechanism of the silver-substituted enzyme is similar to that of the native enzyme. Our results suggest that the Ag-substituted enzyme can oxidize CO and release CO2 following the same reaction steps as the native enzyme, with a computed rate-limiting step of 10.4 kcal/mol, consistent with experimental findings. Surprisingly, lower activation energies for C-O bond formation have been found in the presence of silver. Furthermore, comparison of rate constants for reduction of copper- and silver-containing enzymes suggests a discrepancy in the transition state stabilization upon silver substitution. We also evaluated the effects that differences in the water-active site interaction may exert on the overall energy profile of catalysis. Finally, the formation of a thiocarbonate intermediate along the catalytic pathway was found to be energetically unfavorable for the Ag-substituted enzyme. This finding aligns with the hypothesis proposed for the wild-type form, suggesting that the creation of such species may not be necessary for the enzymatic catalysis of CO oxidation.

GSNOR negatively regulates the NLRP3 inflammasome via S-nitrosation of MAPK14.

Hyperactivation of the NLRP3 inflammasome has been implicated in the pathogenesis of numerous diseases. However, the precise molecular mechanisms that modulate the transcriptional regulation of NLRP3 remain largely unknown. In this study, we demonstrated that S-nitrosoglutathione reductase (GSNOR) deficiency in macrophages leads to significant increases in the Nlrp3 and Il-1ß expression levels and interleukin-1ß (IL-1ß) secretion in response to NLRP3 inflammasome stimulation. Furthermore, in vivo experiments utilizing Gsnor-/- mice revealed increased disease severity in both lipopolysaccharide (LPS)-induced septic shock and dextran sodium sulfate (DSS)-induced colitis models. Additionally, we showed that both LPS-induced septic shock and DSS-induced colitis were ameliorated in Gsnor-/- Nlrp3-/- double-knockout (DKO) mice. Mechanistically, GSNOR deficiency increases the S-nitrosation of mitogen-activated protein kinase 14 (MAPK14) at the Cys211 residue and augments MAPK14 kinase activity, thereby promoting Nlrp3 and Il-1ß transcription and stimulating NLRP3 inflammasome activity. Our findings suggested that GSNOR is a regulator of the NLRP3 inflammasome and that reducing the level of S-nitrosylated MAPK14 may constitute an effective strategy for alleviating diseases associated with NLRP3-mediated inflammation.

Identification of a cinnamoyl-CoA reductase from Cinnamomum cassia involved in trans-cinnamaldehyde biosynthesis.

MAIN CONCLUSION: The identification of a functional cinnamoyl-CoA reductase enzyme from Cinnamomum cassia involved in trans-cinnamaldehyde biosynthesis offers the potential for enhancing trans-cinnamaldehyde production through genetic engineering. A significant accumulation of trans-cinnamaldehyde has been found in the bark tissues of C. cassia, used in traditional Chinese medicine. trans-Cinnamaldehyde exhibits various pharmacological properties such as anti-inflammatory, analgesic, and protection of the stomach and the digestive tract. However, further elucidation and characterization of the biosynthetic pathway for trans-cinnamaldehyde is required. In this study, we conducted an integrated analysis of trans-cinnamaldehyde accumulation profiles and transcriptomic data from five different C. cassia tissues to identify the genes involved in its biosynthesis. The transcriptome data we obtained included nearly all genes associated with the trans-cinnamaldehyde pathway, with the majority demonstrating high abundance in branch barks and trunk barks. We successfully cloned four C. cassia cinnamoyl-CoA reductases (CcCCRs), a key gene in trans-cinnamaldehyde biosynthesis. We found that the recombinant CcCCR1 protein was the only one that more efficiently converted cinnamoyl-CoA into trans-cinnamaldehyde. CcCCR1 exhibited approximately 14.7-fold higher catalytic efficiency (kcat/Km) compared to the Arabidopsis thaliana cinnamoyl-CoA reductase 1 (AtCCR1); therefore, it can be utilized for engineering higher trans-cinnamaldehyde production as previously reported. Molecular docking studies and mutagenesis experiments also validated the superior catalytic activity of CcCCR1 compared to AtCCR1. These findings provide valuable insights for the functional characterization of enzyme-coding genes and hold potential for future engineering of trans-cinnamaldehyde biosynthetic pathways.

Investigating human-derived lactic acid bacteria for alcohol resistance.

BACKGROUND: Excessive alcohol consumption has been consistently linked to serious adverse health effects, particularly affecting the liver. One natural defense against the detrimental impacts of alcohol is provided by alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH), which detoxify harmful alcohol metabolites. Recent studies have shown that certain probiotic strains, notably Lactobacillus spp., possess alcohol resistance and can produce these critical enzymes. Incorporating these probiotics into alcoholic beverages represents a pioneering approach that can potentially mitigate the negative health effects of alcohol while meeting evolving consumer preferences for functional and health-centric products. RESULTS: Five lactic acid bacteria (LAB) isolates were identified: Lactobacillus paracasei Alc1, Lacticaseibacillus rhamnosus AA, Pediococcus acidilactici Alc3, Lactobacillus paracasei Alc4, and Pediococcus acidilactici Alc5. Assessment of their alcohol tolerance, safety, adhesion ability, and immunomodulatory effects identified L. rhamnosus AA as the most promising alcohol-tolerant probiotic strain. This strain also showed high production of ADH and ALDH. Whole genome sequencing analysis revealed that the L. rhamnosus AA genome contained both the adh (encoding for ADH) and the adhE (encoding for ALDH) genes. CONCLUSIONS: L. rhamnosus AA, a novel probiotic candidate, showed notable alcohol resistance and the capability to produce enzymes essential for alcohol metabolism. This strain is a highly promising candidate for integration into commercial alcoholic beverages upon completion of comprehensive safety and functionality evaluations.

Silencing NlFAR7 destroyed the pore canals and related structures of the brown planthopper.

Fatty acyl-CoA reductase (FAR) is one of the key enzymes, which catalyses the conversion of fatty acyl-CoA to the corresponding alcohols. Among the FAR family members in the brown planthopper (Nilaparvata lugens), NlFAR7 plays a pivotal role in both the synthesis of cuticular hydrocarbons and the waterproofing of the cuticle. However, the precise mechanism by which NlFAR7 influences the formation of the cuticle structure in N. lugens remains unclear. Therefore, this paper aims to investigate the impact of NlFAR7 through RNA interference, transmission electron microscope, focused ion beam scanning electron microscopy (FIB-SEM) and lipidomics analysis. FIB-SEM is employed to reconstruct the three-dimensional (3D) architecture of the pore canals and related cuticle structures in N. lugens subjected to dsNlFAR7 and dsGFP treatments, enabling a comprehensive assessment of changes in the cuticle structures. The results reveal a reduction in the thickness of the cuticle and disruptions in the spiral structure of pore canals, accompanied by widened base and middle diameters. Furthermore, the lipidomics comparison analysis between dsNlFAR7- and dsGFP-treated N. lugens demonstrated that there were 25 metabolites involved in cuticular lipid layer synthesis, including 7 triacylglycerols (TGs), 5 phosphatidylcholines (PCs), 3 phosphatidylethanolamines (PEs) and 2 diacylglycerols (DGs) decreased, and 4 triacylglycerols (TGs) and 4 PEs increased. In conclusion, silencing NlFAR7 disrupts the synthesis of overall lipids and destroys the cuticular pore canals and related structures, thereby disrupting the secretion of cuticular lipids, thus affecting the cuticular waterproofing of N. lugens. These findings give significant attention with reference to further biochemical researches on the substrate specificity of FAR protein, and the molecular regulation mechanisms during N. lugens life cycle.