Enzyme Catalyzed Formation of CoA Adducts of Fluorinated Hexanoic Acid Analogues using a Long-Chain acyl-CoA Synthetase from Gordonia sp. Strain NB4-1Y.

Per and polyfluoroalkyl substances (PFAS) are a large family of anthropogenic fluorinated chemicals of increasing environmental concern. Over recent years, numerous microbial communities have been found to be capable of metabolizing some polyfluoroalkyl substances, generating a range of low-molecular-weight PFAS metabolites. One proposed pathway for the microbial breakdown of fluorinated carboxylates includes ß-oxidation, this pathway is initiated by the formation of a CoA adduct. However, until recently no PFAS-CoA adducts had been reported. In a previous study, we were able to use a bacterial medium-chain acyl-CoA synthetase (mACS) to form CoA adducts of fluorinated adducts of propanoic acid and pentanoic acid but were not able to detect any products of fluorinated hexanoic acid analogues. Herein, we expressed and purified a long-chain acyl-CoA synthetase (lACS) and a A461K variant of mACS from the soil bacterium Gordonia sp. strain NB4-1Y and performed an analysis of substrate scope and enzyme kinetics using fluorinated and nonfluorinated carboxylates. We determined that lACS can catalyze the formation of CoA adducts of 1:5 fluorotelomer carboxylic acid (FTCA), 2:4 FTCA and 3:3 FTCA, albeit with generally low turnover rates (<0.02 s-1) compared with the nonfluorinated hexanoic acid (5.39 s-1). In addition, the A461K variant was found to have an 8-fold increase in selectivity toward hexanoic acid compared with wild-type mACS, suggesting that Ala-461 has a mechanistic role in selectivity toward substrate chain length. This provides further evidence to validate the proposed activation step involving the formation of CoA adducts in the enzymatic breakdown of PFAS.

Diethyl aminoethyl hexanoate ameliorates salt tolerance associated with ion transport, osmotic adjustment, and metabolite reprograming in white clover.

BACKGROUND: Soil salinization is a serious environmental hazard, limiting plant growth and production in different agro-ecological zones worldwide. Diethyl aminoethyl hexanoate (DA-6) as an essential plant growth regulator (PGR) exhibits a beneficial role in improving crop growth and stress tolerance. However, the DA-6-regulated effect and mechanism of salt tolerance in plants are still not fully understood. The objective of current study was to disclose salt tolerance induced by DA-6 in relation to changes in water and redox balance, photosynthetic function, ionic homeostasis, and organic metabolites reprogramming in white clover (Trifolium repens). RESULTS: A prolonged duration of salt stress caused water loss, impaired photosynthetic function, and oxidative injury to plants. However, foliar application of DA-6 significantly improved osmotic adjustment (OA), photochemical efficiency, and cell membrane stability under salt stress. In addition, high salinity induced massive accumulation of sodium (Na), but decreased accumulation of potassium (K) in leaves and roots of all plants. DA-6-treated plants demonstrated significantly higher transcript levels of genes involved in uptake and transport of Na and K such as VP1, HKT8, SOS1, NHX2, NHX6, and SKOR in leaves as well as VP1, HKT1, HKT8, H+-ATPase, TPK5, SOS1, NHX2, and SKOR in roots. Metabolomics analysis further illustrated that DA-6 primarily induced the accumulation of glucuronic acid, hexanoic acid, linolenic acid, arachidonic acid, inosose, erythrulose, galactopyranose, talopyranose, urea, 1-monopalmitin, glycerol monostearate, campesterol, stigmasterol, and alanine. CONCLUSIONS: The DA-6 significantly up-regulated transcript levels of multiple genes associated with increased Na+ compartmentalization in vacuoles and Na+ sequestration in roots to reduce Na+ transport to photosynthetic organs, thereby maintaining Na+ homeostasis under salt stress. The accumulation of many organic metabolites induced by the DA-6 could be attributed to enhanced cell wall and membrane structural stability and functionality, OA, antioxidant defense, and downstream signal transduction in leaves under salt stress. The present study provides a deep insight about the synergistic role of DA-6 in salt tolerance of white clover.

Engineering of the Long-Main-Chain Monomer-Incorporating Polyhydroxyalkanoate Synthase PhaCAR for the Biosynthesis of Poly[(R)-3-hydroxybutyrate-co-6-hydroxyhexanoate].

Polyhydroxyalkanoate (PHA) synthases (PhaCs) are useful and versatile tools for the production of aliphatic polyesters. Here, the chimeric PHA synthase PhaCAR was engineered to increase its capacity to incorporate unusual 6-hydroxyhexanoate (6HHx) units. Mutations at positions 149 and 314 in PhaCAR were previously found to increase the incorporation of an analogous natural monomer, 3-hydroxyhexanoate (3HHx). We attempted to repurpose the mutations to produce 6HHx-containing polymers. Site-directed saturation mutants at these positions were applied for P(3HB-co-6HHx) synthesis in Escherichia coli. As a result, the N149D and F314Y mutants effectively increased the 6HHx fraction. Moreover, the pairwise NDFY mutation further increased the 6HHx fraction, which reached 22 mol %. This increase was presumably caused by altered enzyme activity rather than altered expression levels, as assessed based on immunoblot analysis. The glass transition temperature and crystallinity of P(3HB-co-6HHx) decreased as the 6HHx fraction increased.

Transcriptomic profiling of an evolved Yarrowia lipolytica strain: tackling hexanoic acid fermentation to increase lipid production from short-chain fatty acids.

BACKGROUND: Short-chain fatty acids (SCFAs) are cost-effective carbon sources for an affordable production of lipids. Hexanoic acid, the acid with the longest carbon chain in the SCFAs pool, is produced in anaerobic fermentation of organic residues and its use is very challenging, even inhibiting oleaginous yeasts growth. RESULTS: In this investigation, an adaptive laboratory evolution (ALE) was performed to improve Yarrowia lipolytica ACA DC 50109 tolerance to high hexanoic acid concentrations. Following ALE, the transcriptomic analysis revealed several genetic adaptations that improved the assimilation of this carbon source in the evolved strain compared to the wild type (WT). Indeed, the evolved strain presented a high expression of the up-regulated gene YALI0 E16016g, which codes for FAT1 and is related to lipid droplets formation and responsible for mobilizing long-chain acids within the cell. Strikingly, acetic acid and other carbohydrate transporters were over-expressed in the WT strain. CONCLUSIONS: A more tolerant yeast strain able to attain higher lipid content under the presence of high concentrations of hexanoic acid has been obtained. Results provided novel information regarding the assimilation of hexanoic acid in yeasts.

N-terminal truncation of PhaCBP-M-CPF4 and its effect on PHA production.

BACKGROUND: Among the polyhydroxyalkanoate (PHA), poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)] is reported to closely resemble polypropylene and low-density polyethylene. Studies have shown that PHA synthase (PhaC) from mangrove soil (PhaCBP-M-CPF4) is an efficient PhaC for P(3HB-co-3HHx) production and N-termini of PhaCs influence its substrate specificity, dimerization, granule morphology, and molecular weights of PHA produced. This study aims to further improve PhaCBP-M-CPF4 through N-terminal truncation. RESULTS: The N-terminal truncated mutants of PhaCBP-M-CPF4 were constructed based on the information of the predicted secondary and tertiary structures using PSIPRED server and AlphaFold2 program, respectively. The N-terminal truncated PhaCBP-M-CPF4 mutants were evaluated in C. necator mutant PHB-4 based on the cell dry weight, PHA content, 3HHx molar composition, molecular weights, and granule morphology of the PHA granules. The results showed that most transformants harbouring the N-terminal truncated PhaCBP-M-CPF4 showed a reduction in PHA content and cell dry weight except for PhaCBP-M-CPF4 G8. PhaCBP-M-CPF4 G8 and A27 showed an improved weight-average molecular weight (Mw) of PHA produced due to lower expression of the truncated PhaCBP-M-CPF4. Transformants harbouring PhaCBP-M-CPF4 G8, A27, and T74 showed a reduction in the number of granules. PhaCBP-M-CPF4 G8 produced higher Mw PHA in mostly single larger PHA granules with comparable production as the full-length PhaCBP-M-CPF4. CONCLUSION: This research showed that N-terminal truncation had effects on PHA accumulation, substrate specificity, Mw, and granule morphology. This study also showed that N-terminal truncation of the amino acids that did not adopt any secondary structure can be an alternative to improve PhaCs for the production of PHA with higher Mw in mostly single larger granules.

Analysis of ester-producing performance in high-yield ethyl hexanoate yeast and the effect on metabolites in bio- enhanced Daqu, the starter for Baijiu and other traditional fermented foods.

AIMS: Ethyl hexanoate, one of the key flavor compounds in strong-flavor Baijiu. To improve the content of ethyl hexanoate in strong-flavor Baijiu, a functional strain with high yield of ethyl hexanoate was screened and its ester-producing performance was studied. METHODS AND RESULTS: Upon identification, the strain was classified as Candida sp. and designated as ZY002. Under optimal fermentation conditions, the content of ethyl hexanoate synthesized by ZY002 can be as high as 170.56 mg L-1. A fermentation test was carried out using the ZY002 strain bioaugmented Daqu to verify the role of the strain applied to Baijiu brewing. It was found that strain ZY002 could not only improve the moisture and alcohol contents of fermented grains but also diminish the presence of reducing sugar and crude starch. Furthermore, it notably amplified the abundance of flavor compounds. CONCLUSION: In this study, Candida sp. ZY002 with a high yield of ethyl hexanoate provided high-quality strain resources for the actual industrial production of Baijiu.

Pyruvate stimulates transamination of leucine into α-ketoisocaproic acid and supports 3-methylbutanal production by Lactococcus lactis.

AIM: To investigate the effect of pyruvate and glucose on leucine transamination and 3-methylbutanal production by Lactococcus lactis, including the comparison with cells possessing glutamate dehydrogenase (GDH) activity. METHODS AND RESULTS: Lactococcus lactis cells were incubated in chemically defined medium (CDM) with the pH controlled at 5.2 to mimic cheese conditions. Pyruvate supplementation stimulated the production of the key flavour compound 3-methylbutanal by 3-4 times after 72 h of incubation. Concurrently, alanine production increased, demonstrating the involvement of pyruvate in transamination reactions. Glucose-metabolizing cells excreted α-ketoisocaproic acid and produced even 3 times more 3-methylbutanal after 24 h than pyruvate-supplemented cells. Conjugal transfer technique was used to transfer the plasmid pGdh442 carrying the gdh gene encoding for GDH to L. lactis. Introducing GDH did not stimulate the excretion of α-ketoisocaproic acid and the production of 3-methylbutanal. CONCLUSIONS: These results demonstrate that Lactococcus uses pyruvate to transaminate leucine into α-ketoisocaproic acid which supports 3-methylbutanal production. Surprisingly, GDH activity did not stimulate leucine transamination and 3-methylbutanal production.

Development of an efficient yeast platform for cannabigerolic acid biosynthesis.

Cannabinoids are important therapeutical molecules for human ailments, cancer treatment, and SARS-CoV-2. The central cannabinoid, cannabigerolic acid (CBGA), is generated from geranyl pyrophosphate and olivetolic acid by Cannabis sativa prenyltransferase (CsPT4). Despite efforts to engineer microorganisms such as Saccharomyces cerevisiae (S. cerevisiae) for CBGA production, their titers remain suboptimal because of the low conversion of hexanoate into olivetolic acid and the limited activity and stability of the CsPT4. To address the low hexanoate conversion, we eliminated hexanoate consumption by the beta-oxidation pathway and reduced its incorporation into fatty acids. To address CsPT4 limitations, we expanded the endoplasmic reticulum and fused an auxiliary protein to CsPT4. Consequently, the engineered S. cerevisiae chassis showed a marked improvement of 78.64-fold in CBGA production, reaching a titer of 510.32 ± 10.70 mg l-1 from glucose and hexanoate.

Restricted O2 consumption in pea roots induced by hexanoic acid is linked to depletion of Krebs cycle substrates.

Plant roots are exposed to hypoxia in waterlogged soils, and they are further challenged by specific phytotoxins produced by microorganisms in such conditions. One such toxin is hexanoic acid (HxA), which, at toxic levels, causes a strong decline in root O2 consumption. However, the mechanism underlying this process is still unknown. We treated pea (Pisum sativum L.) roots with 20 mM HxA at pH 5.0 and 6.0 for a short time (1 h) and measured leakage of key electrolytes such as metal cations, malate, citrate and nonstructural carbohydrates (NSC). After treatment, mitochondria were isolated to assess their functionality evaluated as electrical potential and O2 consumption rate. HxA treatment resulted in root tissue extrusion of K+ , malate, citrate and NSC, but only the leakage of the organic acids and NSC increased at pH 5.0, concomitantly with the inhibition of O2 consumption. The activity of mitochondria isolated from treated roots was almost unaffected, showing just a slight decrease in oxygen consumption after treatment at pH 5.0. Similar results were obtained by treating the pea roots with another organic acid with a short carbon chain, that is, butyric acid. Based on these results, we propose a model in which HxA, in its undissociated form prevalent at acidic pH, stimulates the efflux of citrate, malate and NSC, which would, in turn, cause starvation of mitochondrial respiratory substrates of the Krebs cycle and a consequent decline in O2 consumption. Cation extrusion would be a compensatory mechanism in order to restore plasma membrane potential.

Metabolite-Based Mutualistic Interaction between Two Novel Clostridial Species from Pit Mud Enhances Butyrate and Caproate Production.

Pit mud microbial consortia play crucial roles in the formation of Chinese strong-flavor baijiu's key flavor-active compounds, especially butyric and caproic acids. Clostridia, one of the abundant bacterial groups in pit mud, were recognized as important butyric and caproic acid producers. Research on the interactions of the pit mud microbial community mainly depends on correlation analysis at present. Interaction between Clostridium and other microorganisms and its involvement in short/medium-chain fatty acid (S/MCFA) metabolism are still unclear. We previously found coculture of two clostridial strains isolated from pit mud, Clostridium fermenticellae JN500901 (C.901) and Novisyntrophococcus fermenticellae JN500902 (N.902), could enhance S/MCFA accumulation. Here, we investigated their underlying interaction mechanism through the combined analysis of phenotype, genome, and transcriptome. Compared to monocultures, coculture of C.901 and N.902 obviously promoted their growth, including shortening the growth lag phase and increasing biomass, and the accumulation of butyric acid and caproic acid. The slight effects of inoculation ratio and continuous passage on the growth and metabolism of coculture indicated the relative stability of their interaction. Transwell coculture and transcriptome analysis showed the interaction between C.901 and N.902 was accomplished by metabolite exchange, i.e., formic acid produced by C.901 activated the Wood-Ljungdahl pathway of N.902, thereby enhancing its production of acetic acid, which was further converted to butyric acid and caproic acid by C.901 through reverse ß-oxidation. This work demonstrates the potential roles of mutually beneficial interspecies interactions in the accumulation of key flavor compounds in pit mud. IMPORTANCE Microbial interactions played crucial roles in influencing the assembly, stability, and function of the microbial community. The metabolites of pit mud microbiota are the key to flavor formation of Chinese strong-flavor baijiu. So far, researches on the interactions of the pit mud microbial community have been mainly based on the correlation analysis of sequencing data, and more work needs to be performed to unveil the complicated interaction patterns. Here, we identified a material exchange-based mutualistic interaction system involving two fatty acid-producing clostridial strains (Clostridium fermenticellae JN500901 and Novisyntrophococcus fermenticellae JN500902) isolated from pit mud and systematically elucidated their interaction mechanism for promoting the production of butyric acid and caproic acid, the key flavor-active compounds of baijiu. Our findings provide a new perspective for understanding the complicated interactions of pit mud microorganisms.

The transplacental transfer efficiency of per- and polyfluoroalkyl substances (PFAS): a first meta-analysis.

Per- and polyfluorinated substances (PFAS), ubiquitously present in the environment and biota, are transferred to the fetus via the placenta. PFAS can be distinguished, among other things, by their different carbon chain lengths and functional groups. The aim of this study was to provide comprehensive evidence on PFAS transfer rates across the human placental barrier by means of a meta-analysis based upon a systematic review. The available literature up to April 2021 was reviewed and transplacental transfer efficiencies (TTEs) of PFAS assessed. A total of 39 studies reporting data on 20 PFAS were included in the systematic review. Of these, 20 studies with data on 19 compounds were included in the meta-analysis. Comprehensive Meta-Analysis (CMA v3.0) was used for quantitative, statistical analyses with random effects models. A curvilinear relationship was found with short and long chains of perfluorocarboxylic acids (PFCAs) exhibiting higher TTE than compounds with intermediate chain length. Among the less well studied PFAS, perfluorohexanoic acid (PFHxA), 6:2 fluorotelomersulfonic acid (6:2 FTS) and perfluorobutanoic acid (PFBA) stood out the most with a high TEEs. The dependence of TTEs on chain length and functional group is clearly shown in this first meta-analysis on PFAS transfer across the human placenta. More data on effects of less well studied PFAS in pregnant women and neonates are needed to assess the potential risk for fetal exposure.

The olfactory coreceptor IR8a governs larval feces-mediated competition avoidance in a hawkmoth.

Finding a suitable oviposition site is a challenging task for a gravid female moth. At the same time, it is of paramount importance considering the limited capability of most caterpillars to relocate to alternative host plants. The hawkmoth, Manduca sexta (Sphingidae), oviposits on solanaceous plants. Larvae hatching on a plant that is already attacked by conspecific caterpillars can face food competition, as well as an increased exposure to predators and induced plant defenses. Here, we show that feces from conspecific caterpillars are sufficient to deter a female M. sexta from ovipositing on a plant and that this deterrence is based on the feces-emitted carboxylic acids 3-methylpentanoic acid and hexanoic acid. Using a combination of genome editing (CRISPR-Cas9), electrophysiological recordings, calcium imaging, and behavioral analyses, we demonstrate that ionotropic receptor 8a (IR8a) is essential for acid-mediated feces avoidance in ovipositing hawkmoths.

Microbial Ecological Mechanism for Long-Term Production of High Concentrations of n-Caproate via Lactate-Driven Chain Elongation.

Lactate-driven chain elongation (LCE) has emerged as a new biotechnology to upgrade organic waste streams into a valuable biochemical and fuel precursor, medium-chain carboxylate, n-caproate. Considering that a low cost of downstream extraction is critical for biorefinery technology, a high concentration of n-caproate production is very important to improve the scale-up of the LCE process. We report here that in a nonsterile open environment, the n-caproate concentration was increased from the previous record of 25.7 g·liter-1 to a new high level of 33.7 g·liter-1 (76.8 g chemical oxygen demand [COD]·liter -1), with the highest production rate being 11.5 g·liter-1·day-1 (26.2 g COD·liter -1·day-1). In addition, the LCE process remained stable, with an average concentration of n-caproate production of 20.2 ± 5.62 g·liter-1 (46.1 ± 12.8 g COD·liter -1) for 780 days. Dynamic changes in taxonomic composition integrated with metagenomic data reveal the microbial ecology for long-term production of high concentrations of n-caproate: (i) the core microbiome is related to efficient functional groups, such as Ruminococcaceae (with functional strain CPB6); (ii) the core bacteria can maintain stability for long-term operation; (iii) the microbial network has relatively low microbe-microbe interaction strength; and (iv) low relative abundance and variety of competitors. The network structure could be shaped by hydraulic retention time (HRT) over time, and long-term operation at an HRT of 8 days displayed higher efficacy.IMPORTANCE Our research revealed the microbial network of the LCE reactor microbiome for n-caproate production at high concentrations, which will provide a foundation for designing or engineering the LCE reactor microbiome to recover n-caproate from organic waste streams in the future. In addition, the hypothetical model of the reactor microbiome that we proposed may offer guidance for researchers to find the underlying microbial mechanism when they encounter low-efficiency n-caproate production from the LCE process. We anticipate that our research will rapidly advance LCE biotechnology with the goal of promoting the sustainable development of human society.

Adaptability of a Caproate-Producing Bacterium Contributes to Its Dominance in an Anaerobic Fermentation System.

The transformation of diverse feedstocks into medium-chain fatty acids (MCFAs) by mixed cultures is a promising biorefinery route because of the high value of MCFAs. A particular concern is how to maintain the microbial consortia in mixed cultures to achieve stable MCFA production. The Chinese strong aroma-type liquor (Baijiu) fermentation system continually produces caproic acid for decades through a spontaneous inoculation of anaerobes from pit mud into fermented grains. Therefore, illuminating the dominant caproate-producing bacterium (CPB) in pit mud and how the CPB is sustained in the spontaneous fermentation system will help to reveal the microbiological mechanisms of stable caproate production. Here, we examined pit mud samples across four Chinese strong aroma-type Baijiu-producing areas and found that a caproate-producing Caproicibacterium sp. was widely distributed in these distilleries, with relative abundance ranging from 1.4 to 35.5% and an average abundance of 11.4%. Through controlling carbon source availability, we obtained different simplified caproate-producing consortia and found that the growth advantage of Caproicibacterium sp. was highly dependent on glucose. Then, two strains, named Caproicibacterium sp. strain LBM19010 and Caproicibacterium sp. strain JNU-WLY1368, were isolated from pit mud of two regions. The metabolic versatility of this species utilizing starch, maltose, glucose, and lactate reflected its adaptability to the fermentation environment where these carbon sources coexist. The simultaneous utilization of glucose and lactate contributed to the balance between cell growth and pH homeostasis. This study reveals that multiple adaptation strategies employed by the predominant CPB promotes its stability and dominance in a saccharide- and lactate-rich anaerobic habitat. IMPORTANCE The Chinese strong aroma-type liquor (Baijiu) fermentation environment is a typical medium-chain fatty acid-producing system with complex nutrients. Although several studies have revealed the correlation between microbial community composition and abiotic factors, the adaptation mechanisms of dominant species to abiotic environment are still unknown in this special anaerobic habitat. This study identified the predominant CPB in Chinese strong aroma-type Baijiu fermentation system. Metabolic versatility and flexibility of the dominant CPB with a small-size genome indicated that this bacterium can effectively exploit available carbon and nitrogen sources, which could be a key factor to promote its ecological success in a multispecies environment. The understanding of growth and metabolic features of the CPB responsible for its dominance in microbial community will not only contribute to the improvement of Chinese strong aroma-type Baijiu production but also expand its potential industrial applications in caproate production.

Discovery of 6-Oxo-4-phenyl-hexanoic acid derivatives as RORγt inverse agonists showing favorable ADME profile.

The retinoic acid receptor-related orphan nuclear receptor gamma t (RORγt), which is a promising therapeutic target for immune diseases, is a major transcription factor of genes related to psoriasis pathogenesis, such as interleukin (IL)-17A, IL-22, and IL-23R. Inspired by the co-crystal structure of RORγt, a 6-oxo-4-phenyl-hexanoic acid derivative 6a was designed, synthesized, and identified as a ligand of RORγt. The structure-activity relationship (SAR) studies in 6a, which focus on the improvement of its membrane permeability profile by introducing chlorine atoms, led to finding 12a, which has a potent RORγt inhibitory activity and a favorable pharmacokinetic profile.

PFAS Degradation in Ultrapure and Groundwater Using Non-Thermal Plasma.

Perfluoroalkyl substances (PFAS) represent one of the most recalcitrant class of compounds of emerging concern and their removal from water is a challenging goal. In this study, we investigated the removal efficiency of three selected PFAS from water, namely, perfluorooctanoic acid (PFOA), perfluorohexanoic acid (PFHxA) and pefluorooctanesulfonic acid (PFOS) using a custom-built non-thermal plasma generator. A modified full factorial design (with 2 levels, 3 variables and the central point in which both quadratic terms and interactions between couple of variables were considered) was used to investigate the effect of plasma discharge frequency, distance between the electrodes and water conductivity on treatment efficiency. Then, the plasma treatment running on optimized conditions was used to degrade PFAS at ppb level both individually and in mixture, in ultrapure and groundwater matrices. PFOS 1 ppb exhibited the best degradation reaching complete removal after 30 min of treatment in both water matrices (first order rate constant 0.107 min-1 in ultrapure water and 0.0633 min-1 in groundwater), while the degradation rate of PFOA and PFHxA was slower of around 65% and 83%, respectively. During plasma treatment, the production of reactive species in the liquid phase (hydroxyl radical, hydrogen peroxide) and in the gas phase (ozone, NOx) was investigated. Particular attention was dedicated to the nitrogen balance in solution where, following to NOx hydrolysis, total nitrogen (TN) was accumulated at the rate of up to 40 mgN L-1 h-1.

Cascade Synthesis from Cyclohexane to ϵ-Caprolactone by Visible-Light-Driven Photocatalysis Combined with Whole-Cell Biological Oxidation.

Cyclohexane was directly oxy-functionalised to ϵ-caprolactone through a cascade reaction sequence combining visible-light-driven photocatalysis with cyclohexanone monooxygenase (CHMO) whole-cell biocatalysis. Two available photocatalysts, Au-doped TiO2 (Au-TiO2 ) and graphitic carbonitride (g-C3 N4 ), were evaluated in the experiment and some optimisations to the cascade reaction were applied. In stepwise mode, the highest degree of conversion from cyclohexanol to ϵ-caprolactone can be up to 41 %, with use of g-C3 N4 . The cascade reaction from cyclohexane to ϵ-caprolactone is achievable under a light intensity of 149 µW cm-2 .

Kartogenin preconditioning commits mesenchymal stem cells to a precartilaginous stage with enhanced chondrogenic potential by modulating JNK and ß-catenin-related pathways.

Cartilage engineering strategies using mesenchymal stem cells (MSCs) could provide preferable solutions to resolve long-segment tracheal defects. However, the drawbacks of widely used chondrogenic protocols containing TGF-ß3, such as inefficiency and unstable cellular phenotype, are problematic. In our research, to optimize the chondrogenic differentiation of human umbilical cord MSCs (hUCMSCs), kartogenin (KGN) preconditioning was performed prior to TGF-ß3 induction. hUCMSCs were preconditioned with 1 µM of KGN for 3 d, sequentially pelleted, and incubated with TGF-ß3 for 28 d. Then, the expression of chondrogenesis- and ossification-related genes was evaluated by immunohistochemistry and RT-PCR. The underlying mechanism governing the beneficial effects of KGN preconditioning was explored by phosphorylated kinase screening and validated in vitro and in vivo using JNK inhibitor (SP600125) and ß-catenin activator (SKL2001). After KGN preconditioning, expression of fibroblast growth factor receptor 3, a marker of precartilaginous stem cells, was up-regulated in hUCMSCs. Furthermore, the KGN-preconditioned hUCMSCs efficiently differentiated into chondrocytes with elevated chondrogenic gene ( SOX9, aggrecan, and collagen II) expression and reduced expression of ossific genes (collagen X and MMP13) compared with hUCMSCs treated with TGF-ß3 only. Phosphokinase screening indicated that the beneficial effects of KGN preconditioning are directly related to an up-regulation of JNK phosphorylation and a suppression of ß-catenin levels. Blocking and activating tests revealed that the prochondrogenic effects of KGN preconditioning was achieved mainly by activating the JNK/Runt-related transcription factor (RUNX)1 pathway, and antiossific effects were imparted by suppressing the ß-catenin/RUNX2 pathway. Eventually, tracheal patches, based on KGN-preconditioned hUCMSCs and TGF-ß3 encapsulated electrospun poly( l-lactic acid-co-ε-caprolactone)/collagen nanofilms, were successfully used for restoring tracheal defects in rabbit models. In summary, KGN preconditioning likely improves the chondrogenic differentiation of hUCMSCs by committing them to a precartilaginous stage with enhanced JNK phosphorylation and suppressed ß-catenin. This novel protocol consisting of KGN preconditioning and subsequent TGF-ß3 induction might be preferable for cartilage engineering strategies using MSCs.-Jing, H., Zhang, X., Gao, M., Luo, K., Fu, W., Yin, M., Wang, W., Zhu, Z., Zheng, J., He, X. Kartogenin preconditioning commits mesenchymal stem cells to a precartilaginous stage with enhanced chondrogenic potential by modulating JNK and ß-catenin-related pathways.

Caproicibacter fermentans gen. nov., sp. nov., a new caproate-producing bacterium and emended description of the genus Caproiciproducens.

A strictly anaerobic bacterial strain designated EA1T was isolated from an enrichment culture inoculated with biogas reactor content. Cells of strain EA1T are spore-forming rods (1-3×0.4-0.8 µm) and stain Gram-negative, albeit they possess a Gram-positive type of cell-wall ultrastructure. Growth of strain EA1T was observed at 30 and 37 °C and within a pH range of pH 5-9. The major components recovered in the fatty acid fraction were C14:0, C16:0, C16:0 DMA (dimethyl acetal) and C16:1 ω7c. Strain EA1T fermented several mono- and disaccharides. Metabolic end products from fructose were acetate, butyrate, caproate and lactate. Furthermore, ethanol, CO2 and H2 were identified as products. The genome consists of a chromosome (3.9 Mbp) with 3797 predicted protein-encoding genes and a G+C content of 51.25 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain EA1T represents a novel taxon within the family Oscillospiraceae. The most closely related type strains of EA1T, based on 16S rRNA gene sequence identity, are Caproiciproducens galactitolivorans BS-1T (94.9 %), [Clostridium] leptum DSM 753T (93.8 %), [Clostridium] sporosphaeroides DSM 1294T (91.7 %) and Ruminococcus bromii ATCC 27255T (91.0 %). Further phenotypic characteristics of strain EA1T differentiate it from related, validly described bacterial species. Strain EA1T represents a novel genus and novel species within the family Oscillospiraceae. The proposed name is Caproicibacter fermentans gen. nov., sp. nov. The type strain is EA1T (DSM 107079T=JCM 33110T).

Revealing the bacterial community profiles during the degradation of acetone, propionic and hexanoic acids-components of wastewater from the Fischer-Tropsch process.

The Fischer-Tropsch (F-T) process for production of fuels is entrenched in several countries' approach to meeting energy demands. However, the clean water deficit associated with the down-stream processes has made it necessary to explore bioremediation methods to ameliorate the consequences of its use. In this study, a consortium of bacteria was utilized for determination of biodegradation and removal rates, based on reduction in chemical oxygen demand of a mixture of acetone, propionic acid and hexanoic acid (APH) (all components of F-T wastewater), at an organic loading of 5 and 9.53 g CODL-1. The individual degradation efficiencies of the F-T components were determined using a gas chromatograph. Further, the bacterial consortia responsible for the degradation of the mixture of APH were determined using metagenomics data derived from next-generation sequencing. The overall chemical oxygen demand removal was found to be 88.8% and 82.3% at organic loading of 5 and 9.53 g CODL-1, respectively. The optimal degradation efficiency of acetone, propionic acid and hexanoic acid over a period of 10 days was found to be 100%, 85% and 75.8%, respectively. The primary microbial communities presumed to be responsible for APH degradation by phyla classification across all samples were found to be Proteobacteria (55-92%), Actinobacteria (5-33%) and Firmicutes (0.08-9%). Overall, the study has demonstrated the importance of aerobic consortia interactions in the degradation of components of the F-T wastewater.