Inoculation of Yarrowia lipolytica promotes the growth of lactic acid bacteria, Debaryomyces udenii and the formation of ethyl esters in sour meat.

Yarrowia lipolytica N12 and A13 with high lipase activity obtained by mutagenesis were inoculated into sour meat, and their effects on physicochemical properties, microbial community succession, free amino acids, and volatile compounds of sour meat were investigated. Inoculation fermentation increased the contents of free amino acids observably, rapidly reduced pH, promoted the accumulation of total acids, decreased 2-thiobarbituric acid reactive substances (TBARS) values. In addition, the addition of Y. lipolytica might contribute to the growth of lactic acid bacteria, Candida spp., and Debaryomyces udenii, which play an important role in production of volatile compounds. It was shown that inoculation promoted the production of esters, aldehydes, and alcohols, especially ethyl esters, giving sour meat a better meat flavor. Besides, it was found that Y. lipolytica A13 had better fermenting property. Sample of A13 group had higher contents of ethyl esters, free amino acids and dominant microorganisms. The results may help to provide new strains for sour meat fermentation.

Fuzheng Huayu recipe inhibits bleomycin-induced pulmonary fibrosis in rats by inhibiting M2 polarization of macrophages via the oxidative phosphorylation pathway.

Fuzheng Huayu recipe (FZHYR) is a Chinese patent medicine for the treatment of fibrosis. The effects of FZHYR on pulmonary fibrosis and macrophage polarization were investigated in vitro. FZHYR inhibited pulmonary inflammation and fibrosis and M2 polarization of macrophages in bleomycin-induced pulmonary fibrosis (BPF) of rat model. Differentially expressed genes were screened by high-throughput mRNA sequencing and GSEA showed that oxidative phosphorylation (OXPHOS) was correlated with BPF. FZHYR inhibited expressions of Ndufa2 and Ndufa6 in lung tissues of BPF rats. These findings suggest that OXPHOS pathway serves as a possible target for pulmonary fibrosis therapy by FZHYR.

Production optimization of food functional factor ergothioneine in wild-type red yeast Rhodotorula mucilaginosa DL-X01.

BACKGROUND: Ergothioneine (EGT) is a high-value food functional factor that cannot be synthesized by humans and other vertebrates, and the low yield limits its application. RESULTS: In this study, the optimal fermentation temperature, fermentation time, initial pH, inoculum age, and inoculation ratio on EGT biosynthesis of Rhodotorula mucilaginosa DL-X01 were optimized. In addition, the effects of three key precursor substances - histidine, methionine, and cysteine - on fungal EGT synthesis were verified. The optimal conditions were further obtained by response surface optimization. The EGT yield of R. mucilaginosa DL-X01 under optimal fermentation conditions reached 64.48 ± 2.30 mg L-1 at shake flask fermentation level. Finally, the yield was increased to 339.08 ± 3.31 mg L-1 (intracellular) by fed-batch fermentation in a 5 L bioreactor. CONCLUSION: To the best of our knowledge, this is the highest EGT yield ever reported in non-recombinant strains. The fermentation strategy described in this study will promote the efficient biosynthesis of EGT in red yeast and its sustainable production in the food industry. © 2024 Society of Chemical Industry.

Self-Assembly of a Series of Carbazole-Based Vinyl-benzoxazole Derivatives in Gel Phases.

A series of carbazole-based vinyl-benzoxazole derivatives have been synthesized in order to verify whether X-ray diffraction (XRD) simulation can give more information about intermolecular stacking in the gel phase. It was found that their gelation capabilities were strongly dependent on the length of the alkyl chain. The compounds with shorter alkyl chains have lower critical gelation concentrations (CGCs) in nonpolar alkane and alcohols with longer carbon chains. On the other hand, compounds with long alkyl chains presented small CGCs in polar methanol. Powder XRD structure solution gave more information about intermolecular stacking than the traditional way of analyzing diffraction peaks to derive approximate molecular stacking patterns. The results verified that gelators had a similar head-to-tail π-stacking between aromatic groups in gel phases although different slipping angles existed. Moreover, ordered stacking between the alkyl chains was also present.

Ergothioneine: new functional factor in fermented foods.

Ergothioneine (EGT) is a high-value natural sulfur-containing amino acid and has been shown to possess extremely potent antioxidant and cytoprotective activities. At present, EGT has been widely used in food, functional food, cosmetics, medicine, and other industries, but its low yield is still an urgent problem to overcome. This review briefly introduced the biological activities and functions of EGT, and expounded its specific applications in food, functional food, cosmetic, and medical industries, introduced and compared the main production methods of EGT and respective biosynthetic pathways in different microorganisms. Furthermore, the use of genetic and metabolic engineering methods to improve EGT production was discussed. In addition, the incorporation of some food-derived EGT-producing strains into fermentation process will allow the EGT to act as a new functional factor in the fermented foods.

Relationship between gut microbiota dysbiosis and immune indicator in children with sepsis.

Sepsis is a life-threatening multiple-organ injury caused by disordered host immune response to microbial infection. However, the correlation between gut microbiota dysbiosis and immune indicators remains unexplored. To address this gap in knowledge, we carried out 16 S rDNA sequencing, analyzed clinical fecal samples from children with sepsis (n = 30) and control children (n = 25), and obtained immune indicators, including T cell subtypes (CD3+, CD3+CD4+, CD3+CD8+, and CD4/CD8), NK cells, cytokines (IL-2, IL-4, IL-6, IL-10, TNF-α and IFN-γ), and immunoglobulin indices (IgA, IgE, IgM and IgG). In addition, we analyzed the correlation between gut microbiota dysbiosis and immune indicators, and evaluated the clinical discriminatory power of discovered bacterial biomarkers. We found that children with sepsis exhibited gut bacterial dysbiosis and low alpha diversity. The Spearman's rank correlation coefficient suggested that Rhodococcus erythropolis had a significantly positive correlation with IFN-γ and CD3+ T cells. Klebsiella pneumoniae and Streptococcus mitis were significantly correlated with NK cells. Bacteroides uniformis was significantly positively correlated with IgM and erythrocyte sedimentation rate, and Eubacterium eligens was significantly positively correlated with IL-4 and CD3+CD8+ T cells. The biomarkers discovered in this study had strong discriminatory power. These changes in the gut microbiome may be closely related to immunologic dysfunction and to the development or exacerbation of sepsis. However, a large sample size is required for verification.

Combined effects of lipase and Lactiplantibacillus plantarum 1-24-LJ on physicochemical property, microbial succession and volatile compounds formation in fermented fish product.

BACKGROUND: Studies have shown that either the addition of starter culture or enzyme can improve fermentation in fish or other products. However, little research has been carried out on the effects of coupling starter cultures with lipase on the microbial community and product quality. Suanzhayu is a Chinese fermented fish product that mainly relies on spontaneous fermentation, resulting in an unstable flavor and quality. The present study investigated the impact of lipase and Lactiplantibacillus plantarum 1-24-LJ on the quality of Suanzhayu. RESULTS: Inoculation decreased pH and 2-thiobarbituric acid reactive substances (TBARS) values, and also helped the dominance of the strain in the ecosystem, whereas lipase addition raised TBARS values and had little effect on pH, water activity (aw ) and microbiota. The addition of lipase and/or Lpb. plantarum increased the content of alcohols, aldehydes, ketones, esters and umami amino acids. The co-additions with the most significant effect and the total contents of volatile compounds (VCs) and free amino acids (FAAs) were 1801.92 g per 100 g and 21 357.05 mg per 100 g, respectively. Former-Lactobacillus was negatively correlated with pH, aw and Prevotella, but positively with VCs (ethyl ester of heptanoic acid, ethyl ester of octanoic acid) and FAAs (Tyr, Phe). Furthermore, adding Lpb. plantarum 1-24-LJ alone or in combination with lipase shortened the fermentation process. CONCLUSION: The present study provides a recommended Suanzhayu process approach for improving product quality and flavor, as well as shortening fermentation time, by adding Lpb. plantarum 1-24-LJ with or without lipase. © 2023 Society of Chemical Industry.

Stereospecific synthesis of S-(-)-trans-verbenol and its antipode by inversion of sterically hindered alcohols.

S-(-)-trans-Verbenol (1) and its antipode, R-(+)-trans-verbenol (1') have been confirmed as the critical pheromone components of bark beetles. Synthesis of these two active secondary alcohols (1 and 1') from commercially available starting materials S-α-pinene and R-α-pinene was reported. The key steps were mainly depended on the effective SN2 stereo-inversion of the hydroxy group of sterically hindered alcohols (3 and 3'), using Mitsunobu reaction or hydrolysis of mesylate ester, alternatively. Our results provide a new and stereo-selectivity way to obtain optically active insect pheromones.

RNAi Efficiency through dsRNA Injection Is Enhanced by Knockdown of dsRNA Nucleases in the Fall Webworm, Hyphantria cunea (Lepidoptera: Arctiidae).

RNA interference (RNAi) technology is a promising approach used in pest control. The efficiency of RNAi varies considerably among different insect species, and growing evidence suggests that degradation of double-stranded RNA (dsRNA) prior to uptake is an important factor that limits RNAi efficiency in insects. Our recent work on fall webworm (Hyphantria cunea), an important invasive pest in China, showed a relatively low silencing efficiency of RNAi through dsRNA injection, which is considered the most feasible dsRNA delivery method for inducing RNAi, and the factors involved in the mechanism remain unknown. Herein, we first detected the dsRNA-degrading activity in the hemolymph and gut content of H. cunea in ex vivo assays and observed rapid degradation of dsRNA, especially in the hemolymph, which was complete within only 10 min. To determine whether dsRNA degradation could contribute to the low effectiveness of RNAi in H. cunea, four dsRNA nuclease (dsRNase) genes, HcdsRNase1, HcdsRNase2, HcdsRNase3, and HcdsRNase4, were identified by homology searching against the H. cunea transcriptome database, and their transcript levels were subsequently investigated in different tissues, developmental stages, and after dsRNA injection. Our results show that HcdsRNases are highly expressed mainly in gut tissues and hemolymph, and the expression of HcdsRNase3 and HcdsRNase4 were significantly upregulated by dsGFP induction. RNAi-of-RNAi studies, using HcCht5 as a reporter gene, demonstrated that silencing HcdsRNase3 and HcdsRNase4 significantly increases RNAi efficacy via dsHcCht5 injection, and co-silencing these two HcdsRNase genes results in a more significant improvement in efficacy. These results confirm that the RNAi efficacy in H. cunea through dsRNA injection is certainly impaired by dsRNase activity, and that blocking HcdsRNases could potentially improve RNAi, providing a reference for related studies on insects where RNAi has low efficiency.

Analysis of carotenoid profile changes and carotenogenic genes transcript levels in Rhodosporidium toruloides mutants from an optimized Agrobacterium tumefaciens-mediated transformation method.

Rhodosporidium toruloides has been reported as a potential biotechnological microorganism to produce carotenoids. The most commonly used molecular and genetic manipulation methods based on Agrobacterium-mediated transformation (ATMT). However, this method was of relatively lower transformation efficiency. In this study, we optimized the ATMT method for R. toruloides on account of the promoter on T-DNA, the ratio of A. tumefaciens to R. toruloides NP11, acetosyringone concentration, cocultivation temperature and time, and a transformation efficiency of 2,369 cells per 105 recipient cells was obtained and was 24 times as that of the previous report. With this optimized method, four redder mutants and four yellower mutants were selected out with torularhodin and ß-carotene production preference, respectively. The highest torularhodin production was 1,638.15 µg/g dry cell weight in A1-13. The yellower mutants were found to divert the metabolic flux from torularhodin and torulene to γ-carotene and ß-carotene, and the proportion of γ-carotene and ß-carotene were all over 92%. TAIL-PCR was carried out to found T-DNA insertion in these mutants, and insertion hotspot was found. RT-qPCR results showed that CTA1 genes in these mutants were closely related to the synthesis of total carotenoids, especially torularhodin, and was a potenial metabolic engineering site in the future.

Effects of salt concentration on the quality of paocai, a fermented vegetable product from China.

BACKGROUND: Paocai is a traditional Chinese fermented vegetable food. As the most important ingredient, salt has crucial effects on the bacterial community and volatile compounds of paocai. To demonstrate the effects of salt on the fermentation of paocai, the bacterial composition and volatile compounds were investigated using high-throughput sequencing and gas chromatography-mass spectrometry (GC-MS). RESULTS: The salt had no significant effects on the bacterial community at the phylum level. Proteobacteria and Bacteroidetes gradually decreased during the fermentation, and Firmicutes gradually increased as the dominant bacteria in the late stage of fermentation. At the genus level, Lactobacillus and Lactococcus gradually increased in relative abundance during the fermentation and became the dominant bacteria in paocai. High salt levels can contribute to the growth of Lactobacillus, which became the dominant genus in paocai. The salt concentration affected the profiles of volatile compounds in paocai after fermentation. A total of 42 volatile components were detected by GC-MS, among which phenols, aldehydes, and nitriles were the main ones. A high salt concentration will increase the volatile compound content, mainly aldehydes and alcohols, and improve the flavor of paocai. At the same time, the electronic tongue analysis also showed that a high salt concentration made a major contribution to the flavor of paocai. CONCLUSIONS: These data are helpful to elucidate the effects of salt on the quality of paocai and contribute to improving the quality and reducing the use of salt. © 2021 Society of Chemical Industry.

Chemical signal interactions of the bark beetle with fungal symbionts, and host/non-host trees.

The symbiosis between the bark beetle (Ips subelongatus) and its fungal symbiont (Endoconidiophora fujiensis) poses a serious threat to larch forests. However, the signaling pathways between these symbiotic partners and their host/non-host trees are not fully understood. Inoculation of the host larch (Larix principis-rupprechtii) with two strains of E. fujiensis induced a rapid and long-term release of monoterpenes. Although the fungi had a level of tolerance to these compounds, many monoterpenes inhibited fungal growth in culture. Moreover, monoterpenes with stronger inhibitory effects on fungal growth exhibited weaker synergistic effects on the attraction of I. subelongatus to aggregation pheromone. Surprisingly, individual isomers of aggregation pheromone components promoted fungal symbiont growth in a culture medium. Non-host volatiles (NHVs) were tested and shown to completely inhibit the growth of fungal symbionts in culture but had no effects on beetle responses to aggregation pheromone, with the exception of (Z)-3-hexen-1-ol. These results reveal convergence and mutualism patterns in the evolution of I. subelongatus and E. fujiensis with respect to host tree volatiles but not in response to NHVs. Ultimately, we put forward a hypothesis that host plants are ecological and evolutionary determinants of bark beetle-fungus symbioses in terms of their complex signaling interactions.

The complete mitochondrial genome of the lipid-producing yeast Rhodotorula toruloides.

Mitochondria are semi-autonomous organelles with their own genome and crucial to cellular material and energy metabolism. Here, we report the complete mitochondrial genome of a lipid-producing basidiomycetous yeast Rhodotorula toruloides NP11. The mitochondrial genome of R. toruloides NP11 was assembled into a circular DNA molecule of 125937bp, encoding 15 proteins, 28 transfer RNAs, 2 ribosomal RNA subunits and 10 open reading frames with unknown function. The G + C content (41%) of the mitochondrial genome is substantially lower than that of the nuclear genome (62%) of R. toruloides NP11. Further reanalysis of the transcriptome data confirmed the transcription of four mitochondrial genes. The comparison of the mitochondrial genomes of R. toruloides NP11 and NBRC0880 revealed a significant genetic divergence. These data can complement our understanding of the genetic background of R. toruloides and provide fundamental information for further genetic engineering of this strain.

Rhodosporidium toruloides - A potential red yeast chassis for lipids and beyond.

The red yeast Rhodosporidium toruloides naturally produces microbial lipids and carotenoids. In the past decade or so, many studies demonstrated R. toruloides as a promising platform for lipid production owing to its diverse substrate appetites, robust stress resistance and other favorable features. Also, significant progresses have been made in genome sequencing, multi-omic analysis and genome-scale modeling, thus illuminating the molecular basis behind its physiology, metabolism and response to environmental stresses. At the same time, genetic parts and tools are continuously being developed to manipulate this distinctive organism. Engineered R. toruloides strains are emerging for enhanced production of conventional lipids, functional lipids as well as other interesting metabolites. This review updates those progresses and highlights future directions for advanced biotechnological applications.

Identification of miRNAs and their target genes in Larix olgensis and verified of differential expression miRNAs.

BACKGROUND: MiRNAs (microRNA) are 18-24 nt endogenous noncoding RNAs that regulate gene expression at the post-transcriptional level, including tissue-specific, developmental timing and evolutionary conservation gene expression. RESULTS: This study used high-throughput sequencing technology for the first time in Larix olgensis, predicted 78 miRNAs, including 12,229,003 reads sRNA, screened differentially expressed miRNAs. Predicting target genes was helpful for understanding the miRNA regulation function and obtained 333 corresponding target genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation were analysed, mostly including nucleic acid binding, plant hormone signal transduction, pantothenate and CoA biosynthesis, and cellulose synthase. This study will lay the foundation for clarifying the complex miRNA-mediated regulatory network for growth and development. In view of this, spatio-temporal expression of miR396, miR950, miR164, miR166 and miR160 were analysed in Larix olgensis during the growth stages of not lignified, beginning of lignification, and completely lignified in different tissues (root, stem, and leaf) by quantitative real-time PCR (qRT-PCR). There were differences in the expression of miRNAs in roots, stems and leaves in the same growth period. At 60 days, miR160, miR166 and miR396-2 exhibited the highest expression in leaves. At 120 days, most miRNAs in roots and stems decreased significantly. At 180 days, miRNAs were abundantly expressed in roots and stems. Meanwhile, analysis of the expression of miRNAs in leaves revealed that miR396-2 was reduced as time went on, whereas other miRNAs increased initially and then decreased. On the other hand, in the stems, miR166-1 was increase, whereas other miRNAs, especially miR160, miR164, miR396 and miR950-1, first decreased and then increased. Similarly, in the roots, miR950-2 first decreased and then increased, whereas other miRNAs exhibited a trend of continuous increase. CONCLUSIONS: The present investigation included rapid isolation and identification of miRNAs in Larix olgensis through construction of a sRNA library using Solexa and predicted 78 novel miRNAs, which showed differential expression levels in different tissues and stages. These results provided a theoretical basis for further revealing the genetic regulation mechanism of miRNA in the growth and development of conifers and the verification of function in target genes.

RNA interference in the oleaginous yeast Rhodosporidium toruloides.

The red yeast Rhodosporidium toruloides is an excellent microbial host for production of carotenoids, neutral lipids and valuable enzymes. In recent years, genetic tools for gene expression and gene disruption have been developed for this red yeast. However, methods remain limited in terms of fine-tuning gene expression. In this study, we first demonstrated successful implementation of RNA interference (RNAi) in R. toruloides NP11, which was applied to down-regulate the expression of autophagy related gene 8 (ATG8), and fatty acid synthase genes (FAS1 and FAS2), respectively. Compared with the control strain, RNAi-engineered strains showed a silencing efficiency ranging from 11% to 92%. The RNAi approach described here ensures selective inhibition of the target gene expression, and should expand our capacity in the genetic manipulation of R. toruloides for both fundamental research and advanced cell factory development.

Enhanced resistive acetone sensing by using hollow spherical composites prepared from MoO3 and In2O3.

Hollow sphere composites were synthesized by a template-free hydrothermal method from MoO3 and In2O3. The spheres have a typical size of 800 ± 50 nm and were characterized by XRD, FESEM, TEM, XPS. Gas sensors based on samples with different Mo/In composite ratios were fabricated and their gas sensing properties were studied. The results show that a Mo:In ratio of 1:1 in the composite gives the highest response, typically at a working temperature of 250 °C. The response increases to 38 when exposed to 100 ppm acetone at 250 °C. This is 13.6 times better than when using pure MoO3. The sensor shows improved selectivity, response, repeatability and long-term stability. Typical features include a large specific surface area, and high levels of chemisorbed oxygen and defective oxygen sites. The N-N heterojunction theory was used to explain the improvement of gas sensing performance. Graphical abstract Schematic presentation of MoO3 and In2O3 composites and response test graph for 100 ppm acetone. The sensor based on this composite exhibits a very high response (38) to acetone at 250 °C and very fast response time (2 s).

Facile and Efficient Syntheses of (11Z,13Z)-Hexadecadienal and Its Derivatives: Key Sex Pheromone and Attractant Components of Notodontidae.

Syntheses of (11Z,13Z)-hexadecadienal (1), (11Z,13Z)-hexadecadienol (2), (11Z,13Z)-hexadecadien-1-yl acetate (3), and (Z)-13-hexadecen-11-ynal (4) from commercially available starting material 10-bromo-1-decanol are reported. These (Z,Z)-dienes and conjugated en-yne moieties are common in sex pheromone and attractant components for many Notodontide insect pests. The synthetic scheme, using the C10 + C3 + C3 strategy, was mainly based on three key steps: alkylation of lithium alkyne under a low temperature, cis-Wittig olefination of the aldehyde with propylidentriphenylphosphorane, and hydroboration-protonolysis of alkyne. This synthetic route provided (11Z,13Z)-hexadecadienal (1) in a 23.0% total yield via an eight-step sequence, alcohol (2) in a 21.9% total yield, acetate (3) in a 21.4% total yield, and (Z)-13-hexadecen-11-ynal (4) in a 34.7% total yield. This simple strategy provides a new way to achieve syntheses of the key sex pheromones of Notodontide insect pests.

Efficient co-expression of multiple enzymes from a single promoter mediated by virus 2A sequence in the oleaginous yeast Rhodosporidium toruloides.

The red yeast Rhodosporidium toruloide is a versatile host for production of lipids and carotenoids. Genetic tools are underdeveloped for red yeasts due to their unique genetics and physiology. Currently expression of a heterogonous gene in red yeasts is largely based on integration of the designed cassette by Agrobacterium mediated transformation, yet this method is somewhat restricted when multiple genes are required to be expressed due to the lack of functional genetic elements. Here we demonstrate that virus 2A sequence is effective to mediate co-expression of multiple enzymes in R. toruloides. Two different 2A sequences, Porcine teschovirus-1 2A (P2A) and foot-and-mouth disease virus 2A (F2A), were evaluated. It was found that P2A sequence was more effective for co-expression of two antibiotic selection markers. Co-expression of three antibiotic resistance proteins was successful from a single promoter mediated by P2A sequence. When three heterogeneous enzymes responsible for ß-carotene biosynthesis were co-expressed, recombinant R. toruloides strains produced up to 4.5-fold more ß-carotene than that of the parental one. The use of 2A sequence can facilitate cassette construction to engineer advanced cell factories for production of lipids and related oleochemicals.

Rapid screening of anti-tumor metastasis drugs targeting integrin macrophage antigen-1 using immobilized cell capillary electrophoresis.

In this research a method called immobilized cell capillary electrophoresis (ICCE) was established under approximately physiological conditions for rapid screening of anti-tumor metastasis drugs targeting integrin macrophage antigen-1 (MAC-1). In this method, separation and purification of the target receptors on cell membranes was unnecessary, thus, maintaining their natural conformation and bioactivity. MAC-1-, CD11b-, or CD18-overexpressing HEK293 cells (human embryonic kidney) were cultured and immobilized on the inner wall of capillaries as stationary phase, and their interactions with lactosyl derivative Gu-4 (positive control)/dimethylsulfoxide (DMSO; negative control) were studied using ICCE. Using this method, 29 phenylethanoid glycosides from Cistanches Herba were screened, and the binding kinetic parameters (K, ka, kd, and k') of active compounds were calculated, and the specific subunits of MAC-1 were determined. Then, molecular docking studies were performed to discover the direct interaction sites between active compounds and MAC-1, and the order of Glide-calculated Emodel value obtained from the molecular docking study is consistent with that of the binding constants obtained using ICCE. Finally, pharmaceutical efficacy assays in vitro and in vivo were carried out to show that the anti-tumor metastasis activity of the active compound had better pharmaceutical efficacy and lower toxic side effects. The method was verified to be valid and practical for further use, and it is expected that it will be transferred to capillary array electrophoresis for use in high-throughput drug screening.