Nuclear-targeted chimeric peptide nanorods to amplify innate anti-tumor immunity through localized DNA damage and STING activation.

Stimulator of the interferon genes (STING) pathway is appealing but challenging to potentiate the innate anti-tumor immunity. In this work, nuclear-targeted chimeric peptide nanorods (designated as PFPD) are constructed to amplify innate immunity through localized DNA damage and STING activation. Among which, the chimeric peptide (PpIX-FFVLKPKKKRKV) is fabricated with photosensitizer and nucleus targeting peptide sequence, which can self-assemble into nanorods and load STING agonist of DMXAA. The uniform nanosize distribution and good stability of PFPD improve the sequential targeting delivery of drugs towards tumor cells and nuclei. Under light irradiation, PFPD produce a large amount of reactive oxygen species (ROS) to destroy nuclear DNA in situ, and the released cytosolic DNA fragment will efficiently activate innate anti-tumor immunity in combination with STING agonist. In vitro and in vivo results indicate the superior ability of PFPD to activate natural killer cells and T cells, thus efficiently eradicating lung metastatic tumor without inducing unwanted side effects. This work provides a sophisticated strategy for localized activation of innate immunity for systemic tumor treatment, which may inspire the rational design of nanomedicine for tumor precision therapy.

[Metagenomics unveils the differences in the functions of microbial community of medium-temperature Daqu before and after maturation].

Daqu is the saccharifying, fermenting, and aroma-producing agent used in Baijiu brewing, and its maturation is crucial for obtaining high-quality Daqu. Previous studies have explored the microbial community composition and diversity before and after maturation. However, little is known about the changes in the functions of microbial community. In this study, based on the analyses of enzyme activities and volatile compounds of medium-temperature Daqu before and after maturation, metagenomics was used to analyze the differences in the composition of microbial community and the potential functions, with the aim to explore the microorganisms involved in changes in enzyme activities and important volatiles. The results showed that the moisture (P≤0.05), starch content, liquefying activity, saccharifying activity (P≤0.05), and fermentative activity decreased, while the acidity and esterifying activity (P≤0.05) increased after Daqu maturation. In the meantime, the composition of volatile compounds changed significantly (P=0.001), with significant decreases in the contents of aromatic alcohols and esters as well as significant increases in the contents of pyrazines, ketones, and higher fatty alcohols. The relative abundances of Mucorales (34.8%-23.0%) and Eurotiales (34.3%-20.1%) decreased in matured Daqu, and functional predictions showed these changes decreased the gene abundances of α-amylase, α-glucosidase, alcohol dehydrogenase, and alcohol dehydrogenase (NADP+) (P > 0.05), resulting in lower levels of liquefying activity (P > 0.05), saccharifying activity (P≤0.05), fermentative activity (P > 0.05), as well as aromatic alcohols such as phenylethyl alcohol (P≤0.05). In addition, higher relative abundances of Saccharomycetales (2.9%-16.6%), Lactobacillales (14.9%-23.6%), and Bacillales (0.8%-3.8%) were observed after maturation, and they were conducive to improving the gene abundances of alcohol O-acetyltransferase, carboxylesterase, acetolactate decarboxylase, (R)-acetoin dehydrogenase, and (S)-acetoin dehydrogenase (P≤0.05), resulting in significantly higher levels of esterifying activity and pyrazines (P≤0.05). The microorganisms involved in the changes in enzyme activities and important volatiles before and after Daqu maturation were studied at the gene level in this work, which may facilitate further rational regulation for Daqu production.

Breaking Physical Barrier of Fibrotic Breast Cancer for Photodynamic Immunotherapy by Remodeling Tumor Extracellular Matrix and Reprogramming Cancer-Associated Fibroblasts.

Cancer-associated fibroblasts (CAFs) assist in breast cancer (BRCA) invasion and immune resistance by overproduction of extracellular matrix (ECM). Herein, we develop FPC@S, a photodynamic immunomodulator that targets the ECM, to improve the photodynamic immunotherapy for fibrotic BRCA. FPC@S combines a tumor ECM-targeting peptide, a photosensitizer (protoporphyrin IX) and an antifibrotic drug (SIS3). After anchoring to the ECM, FPC@S causes ECM remodeling and BRCA cell death by generating reactive oxygen species (ROS) in situ. Interestingly, the ROS-mediated ECM remodeling can normalize the tumor blood vessel to improve hypoxia and in turn facilitate more ROS production. Besides, upon the acidic tumor microenvironment, FPC@S will release SIS3 for reprograming CAFs to reduce their activity but not kill them, thus inhibiting fibrosis while preventing BRCA metastasis. The natural physical barrier formed by the dense ECM is consequently eliminated in fibrotic BRCA, allowing the drugs and immune cells to penetrate deep into tumors and have better efficacy. Furthermore, FPC@S can stimulate the immune system and effectively suppress primary, distant and metastatic tumors by combining with immune checkpoint blockade therapy. This study provides different insights for the development of fibrotic tumor targeted delivery systems and exploration of synergistic immunotherapeutic mechanisms against aggressive BRCA.

New perspectives on the genetic structure of dotted gizzard shad (Konosirus punctatus) based on RAD-seq.

To maintain, develop and rationally utilize marine organisms, understanding their genetic structure and habitat adaptation pattern is necessary. Konosirus punctatus, which is a commercial fish species inhabiting the Indo-west Pacific Ocean, has shown an obvious annual global capture and aquaculture production decline due to climate changes and human activities. In the present study, restriction-site associated DNA sequencing (RAD-seq) was used to describe its genome-wide single nucleotide polymorphisms panel (SNPs). Among 146 individuals collected at nine locations scattered in China, Korea and Japan, a set of 632,090 SNPs were identified. Population genetic analysis showed that K. punctatus individuals were divided into two significant genetic clusters. Meanwhile, potential genetic differentiation between northern and southern population of K. punctatus was found. Treemix results indicated that gene flow existed among sampling locations of K. punctatus, especially from southern Japan to others. Moreover, candidate genes associated with habitat adaptations of K. punctatus were identified, which are involved in diverse physiological processes of K. punctatus including growth and development (e.g., KIDINS220, PAN3), substance metabolism (e.g., PGM5) and immune response (e.g., VAV3, CCT7, HSPA12B). Our findings may aid in understanding the possible mechanisms for the population genetic structure and local adaptation of K. punctatus, which is beneficial to establish the management and conservation units of K. punctatus, guiding the rational use of resources, with reference significance for a profound understanding of the adaptative mechanisms of other marine organisms to the environment. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-024-00216-2.

Rapid detection of six Oceanobacillus species in Daqu starter using single-cell Raman spectroscopy combined with machine learning.

Many traditional fermented foods and beverages industries around the world request the addition of multi-species starter cultures. However, the microbial community in starter cultures is subject to fluctuations due to their exposure to an open environment during fermentation. A rapid detection approach to identify the microbial composition of starter culture is essential to ensure the quality of the final products. Here, we applied single-cell Raman spectroscopy (SCRS) combined with machine learning to monitor Oceanobacillus species in Daqu starter, which plays crucial roles in the process of Chinese baijiu. First, a total of six Oceanobacillus species (O. caeni, O. kimchii, O. iheyensis, O. sojae, O. oncorhynchi subsp. Oncorhynchi and O. profundus) were detected in 44 Daqu samples by amplicon sequencing and isolated by pure culture. Then, we created a reference database of these Oceanobacillus strains which correlated their taxonomic data and single-cell Raman spectra (SCRS). Based on the SCRS dataset, five machine-learning algorithms were used to classify Oceanobacillus strains, among which support vector machine (SVM) showed the highest rate of accuracy. For validation of SVM-based model, we employed a synthetic microbial community composed of varying proportions of Oceanobacillus species and demonstrated a remarkable accuracy, with a mean error was less than 1% between the predicted result and the expected value. The relative abundance of six different Oceanobacillus species during Daqu fermentation was predicted within 60 min using this method, and the reliability of the method was proved by correlating the Raman spectrum with the amplicon sequencing profiles by partial least squares regression. Our study provides a rapid, non-destructive and label-free approach for rapid identification of Oceanobacillus species in Daqu starter culture, contributing to real-time monitoring of fermentation process and ensuring high-quality products.

Chromosome-level genome assembly and annotation of eel goby (Odontamblyopus rebecca).

The eel gobies fascinate researchers with many important features, including its unique body structure, benthic lifestyle, and degenerated eyes. However, genome assembly and exploration of the unique genomic composition of the eel gobies are still in their infancy. This has severely limited research progress on gobies. In this study, multi-platform sequencing data were generated and used to assemble and annotate the genome of O. rebecca at the chromosome-level. The assembled genome size of O. rebecca is 918.57 Mbp, which is similar to the estimated genome size (903.03 Mbp) using 17-mer. The scaffold N50 is 41.67 Mbp, and 23 chromosomes were assembled using Hi-C technology with a mounting rate of 99.96%. Genome annotation indicates that 53.29% of the genome is repetitive sequences, and 22,999 protein-coding genes are predicted, of which 21,855 have functional annotations. The chromosome-level genome of O. rebecca will not only provide important genomic resources for comparative genomic studies of gobies, but also expand our knowledge of the genetic origin of their unique features fascinating researchers for decades.

Identification, functional characterization and expression pattern of interferon-gamma (IFN-γ) and interferon-gamma receptor 1 (IFNGR1) in Nibea albiflora.

Interferon-gamma (IFN-γ) is an inflammatory cytokine that plays a crucial role in regulating both innate and cell-mediated immune responses by binding to a receptor complex made up of IFNGR1 and IFNGR2. In this study, the complete cDNA of IFN-γ and IFNGR1 from Nibea albiflora were cloned and functionally characterized (named NaIFN-γ and NaIFNGR1), whose complete cDNA sequences were 1593 bp and 2792 bp, encoding 201 and 399 amino acids, respectively. Multiple sequence alignment and phylogenetic analysis showed that the concluded amino acids sequences of NaIFN-γ and NaIFNGR1 shared high identity with their teleost orthologues including the IFN-γ signature and nuclear localization signal (NLS) motif in NaIFN-γ and FN Ⅲ domain in NaIFNGR1. Real-time PCR showed that NaIFN-γ and NaIFNGR1 constitutively expressed in all tested tissues, such as the head-kidney, spleen, liver, kidney, gill, muscle, blood, and intestine with the highest expression of NaIFN-γ and NaIFNGR1 appearing in the liver and gill, respectively. After experiencing stimulation with Polyinosinic-polycytidylic acid (Poly (I:C)), Vibrio alginolyticus (V. alginolyticus) or Vibrio parahaemolyticus (V. parahaemolyticus), NaIFN-γ and NaIFNGR1 mRNA were up-regulated with the time-dependent model. Due to the presence of a nuclear localization signal (NLS), the subcellular localization revealed that NaIFN-γ dispersed throughout the cytoplasm and nucleus. NaIFNGR1, as a member of Cytokine receptor family B, was primarily expressed on the cell membrane. When NaIFN-γ and NaIFNGR1 were co-transfected, their fluorescence signals overlapped on the membrane of HEK 293T cells indicating the potential interaction between IFN-γ and IFNGR1. The GST-pull-down results further showed that NaIFN-γ could directly interact with the extracellular region of NaIFNGR1, further confirming the affinity between IFN-γ and IFNGR1. Taken together, the results firstly demonstrated that the NaIFN-γ ligand-receptor system existed in N.albiflora and played a pivotal part in N.albiflora's immune response against pathogenic bacterial infections, which contributed to the better understanding of the role of IFN-γ in the immunomodulatory mechanisms of teleost.

Comparative Transcriptomic Analyses Propose the Molecular Regulatory Mechanisms Underlying 1,8-Cineole from Cinnamomum kanehirae Hay and Promote the Asexual Sporulation of Antrodia cinnamomea in Submerged Fermentation.

Antrodia cinnamomea is a valuable edible and medicinal mushroom with antitumor, hepatoprotective, and antiviral effects that play a role in intestinal flora regulation. Spore-inoculation submerged fermentation has become the most efficient and well-known artificial culture process for A. cinnamomea. In this study, a specific low-molecular compound named 1,8-cineole (cineole) from Cinnamomum kanehirae Hay was first reported to have remarkably promoted the asexual sporulation of A. cinnamomea in submerged fermentation (AcSmF). Then, RNA sequencing, real-time quantitative PCR, and a literature review were performed to predict the molecular regulatory mechanisms underlying the cineole-promoted sporulation of AcSmF. The available evidence supports the hypothesis that after receiving the signal of cineole through cell receptors Wsc1 and Mid2, Pkc1 promoted the expression levels of rlm1 and wetA and facilitated their transfer to the cell wall integrity (CWI) signal pathway, and wetA in turn promoted the sporulation of AcSmF. Moreover, cineole changed the membrane functional state of the A. cinnamomea cell and thus activated the heat stress response by the CWI pathway. Then, heat shock protein 90 and its chaperone Cdc37 promoted the expression of stuA and brlA, thus promoting sporulation of AcSmF. In addition, cineole promoted the expression of areA, flbA, and flbD through the transcription factor NCP1 and inhibited the expression of pkaA through the ammonium permease of MEP, finally promoting the sporulation of AcSmF. This study may improve the efficiency of the inoculum (spores) preparation of AcSmF and thereby enhance the production benefits of A. cinnamomea.

Self-reinforced photodynamic immunostimulator to downregulate and block PD-L1 for metastatic breast cancer treatment.

Tumor cells overexpress programmed cell death ligand 1 (PD-L1) to impede immune responses and escape immune elimination. Development of effective combination regimens to sensitize immunotherapy is promising but always challenging. Herein, a self-reinforced photodynamic immunostimulator (designated as PCS) is constructed for metastatic breast cancer treatment through simultaneous downregulation and blockade of PD-L1. Specifically, PCS is prepared by encapsulating signal transducer and activator of transcription 3 (STAT3) inhibitor (Stattic) into photosensitizer (protoporphyrin IX) modified PD-L1 blockade peptide (CVRARTR) through drug self-assembly. PCS can facilitate the targeted drug accumulation in PD-L1 overexpressed breast cancer cells to block PD-L1 and inhibit the phosphorylation of STAT3 to downregulate PD-L1. Moreover, PCS increases intracellular oxidative stress to show a robust anti-proliferation effect through photodynamic therapy (PDT), which also triggers an immunogenic cell death (ICD) to expose the immunostimulatory signals. Consequently, the efficient PD-L1 inhibition and robust PDT of PCS synergistically suppress the malignant growth of breast cancer, and concurrently activate the systemic anti-tumor immunity for metastatic inhibition with no obvious side effects. Such a photodynamic immunostimulator may provide an effective combination regimen for therapies activated immunotherapy against metastatic breast cancer.

Solibacillus daqui sp. nov., isolated from high-temperature Daqu.

A Gram-stain-positive, rod-shaped, endospore-forming, aerobic bacterial strain, designated ZS111008T, was isolated from high-temperature Daqu, a starter for production of Chinese Jiang-flavour Baijiu, and was characterized by polyphasic taxonomy. This novel isolate grew in the presence of 0-5 % (w/v) NaCl, at pH 6.0-9.0 and 25-45 °C; optimum growth was observed with 1 % (w/v) NaCl, at pH 8.0 and 30 °C. A comparative analysis of the 16S rRNA gene sequence (1461 bp) of strain ZS111008T showed highest similarity to Solibacillus silvestris DSM12223T (96.7%), followed by Solibacillus cecembensis PN5T (96.6%) and Solibacillus isronensis AMCK01000046 (96.5%). The DNA G+C content of strain ZS111008T was 37.21 mol%. The respiratory quinone was identified as menaquinone-7 and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine and one unknown phospholipid. Lys was detected as the diagnostic diamino acid in the cell wall. Based on morphological characteristics, chemotaxonomic characteristics and physiological properties, strain ZS111008T represents a novel species of the genus Solibacillus, for which the name Solibacillus daqui sp. nov. is proposed. The type strain for this proposed species is ZS111008T (=CGMCC 1.19455T=JCM 35214T).

Climate Adaptation and Drift Shape the Genomes of Two Eel-Goby Sister Species Endemic to Contrasting Latitude.

Deciphering the role of climate adaptation in generating genetic divergence and hence speciation is a central question in evolution. Comparisons of genomes of closely related species spanning selective climate gradients are particularly informative in discerning the signatures of selection and thereby providing valuable information concerning the role of climate adaptation in speciation. Here we re-sequenced 99 genomes of the two sister eel-goby species Odontamblyopus lacepedii and O. rebecca, which are endemic to tidal mudflats spanning contrasting latitude gradients, to estimate the influence of divergent climate selection on shaping genome-wide patterns of divergence. The results indicated that genome-wide differentiation between the two species was evident (genome-wide FST = 0.313). Against a background of high baseline genomic divergence, 588 and 1202 elevated divergent loci were detected to be widespread throughout their genomes, as opposed to focused within small islands of genomic regions. These patterns of divergence may arise from divergent climate selection in addition to genetic drift acting through past glacial segregation (1.46 million years ago). We identified several candidate genes that exhibited elevated divergence between the two species, including genes associated with substance metabolism, energy production, and response to environmental cues, all putative candidates closely linked to thermal adaptation expected from the latitude gradient. Interestingly, several candidates related to gamete recognition and time of puberty, and also exhibited elevated divergence, indicating their possible role in pre-zygote isolation and speciation of the two species. Our results would expand our knowledge on the roles of latitude climate adaptation and genetic drift in generating and maintaining biodiversity in marine teleosts.

Microbial biogeography of pit mud from an artificial brewing ecosystem on a large time scale: all roads lead to Rome.

IMPORTANCE: Baijiu is a typical example of how humans employ microorganisms to convert grains into new flavors. Mud cellars are used as the fermentation vessel for strong-flavor Baijiu (SFB) to complete the decomposition process of grains. The typical flavor of SFB is mainly attributed to the metabolites of the pit mud microbiome. China has a large number of SFB-producing regions. Previous research revealed the temporal profiles of the pit mud microbiome in different geographical regions. However, each single independent study rarely yields a thorough understanding of the pit mud ecosystem. Will the pit mud microbial communities in different production regions exhibit similar succession patterns and structures under the impact of the brewing environment? Hence, we conducted research in pit mud microbial biogeography to uncover the impact of specific environment on the microbial community over a long time scale.

Genome Sequencing Provides Novel Insights into Mudflat Burrowing Adaptations in Eel Goby Taenioides sp. (Teleost: Amblyopinae).

Amblyopinae is one of the lineage of bony fish that preserves amphibious traits living in tidal mudflat habitats. In contrast to other active amphibious fish, Amblyopinae species adopt a seemly more passive lifestyle by living in deep burrows of mudflat to circumvent the typical negative effects associated with terrestriality. However, little is known about the genetic origin of these mudflat deep-burrowing adaptations in Amblyopinae. Here we sequenced the first genome of Amblyopinae species, Taenioides sp., to elucidate their mudflat deep-burrowing adaptations. Our results revealed an assembled genome size of 774.06 Mb with 23 pseudochromosomes anchored, which predicted 22,399 protein-coding genes. Phylogenetic analyses indicated that Taenioides sp. diverged from the active amphibious fish of mudskipper approximately 28.3 Ma ago. In addition, 185 and 977 putative gene families were identified to be under expansion, contraction and 172 genes were undergone positive selection in Taenioides sp., respectively. Enrichment categories of top candidate genes under significant expansion and selection were mainly associated with hematopoiesis or angiogenesis, DNA repairs and the immune response, possibly suggesting their involvement in the adaptation to the hypoxia and diverse pathogens typically observed in mudflat burrowing environments. Some carbohydrate/lipid metabolism, and insulin signaling genes were also remarkably alterated, illustrating physiological remolding associated with nutrient-limited subterranean environments. Interestingly, several genes related to visual perception (e.g., crystallins) have undergone apparent gene losses, pointing to their role in the small vestigial eyes development in Taenioides sp. Our work provide valuable resources for understanding the molecular mechanisms underlying mudflat deep-burrowing adaptations in Amblyopinae, as well as in other tidal burrowing teleosts.

Lentibacillus daqui sp. nov., isolated from high-temperature Daqu, a starter for production of Chinese Jiang-flavour Baijiu.

A Gram-stain-positive, rod-shaped, endospore-forming, aerobic bacterial strain, designated ZS110521T, was isolated from high-temperature Daqu, a starter for production of Chinese Jiang-flavour Baijiu and was characterised by polyphasic taxonomy. This novel isolate grew in the presence of 0-20 % (w/v) NaCl, at pH 6.0-9.0 and 20-50 °C; optimum growth was observed with 8-10 % (w/v) NaCl, at pH 7.0 and 37 °C. A comparative analysis of the 16S rRNA gene sequence (1460 bp) of ZS110521T revealed that it displayed the highest similarity to Lentibacillus populi WD4L-1T (95.5 %), followed by Lentibacillus garicola SL-MJ1T (95.4 %) and Lentibacillus lacisalsi BH260T (95.2 %). ANI and dDDH values between ZS110521T and other strains of species of the genus Lentibacillus were less than 78 and 28 %, respectively. The predominant cellular fatty acids (> 10 %) of ZS110521T were anteiso-C17 : 0 (37.8 %), anteiso-C15 : 0 (28.1 %) and iso-C16 : 0 (15.5 %). The respiratory quinone was identified as menaquinone-7 (MK-7) and the major polar lipids were diphosphatidylglycerol and phosphatidylglycerol. The polyphasic taxonomic data and the results of chemotaxonomic analysis confirmed that ZS110521T represents a novel species, for which the name Lentibacillus daqui sp. nov. is proposed. The type strain of this proposed species is ZS110521T (=CGMCC 1.19456T =JCM 35213T).

Chromosome-level genome assembly and population genomic analysis reveal evolution and local adaptation in common hairfin anchovy (Setipinna tenuifilis).

Understanding the genetic structure and the factors associated with adaptive diversity has significant implications for the effective management of wild populations under threat from overfishing and climate change. The common hairfin anchovy (Setipinna tenuifilis) is an economically and ecologically important pelagic fish species, spanning a broad latitudinal gradient along marginal seas of the Northwest Pacific. In this study, we constructed the first reference genome of S. tenuifilis using PacBio long reads and high-resolution chromosome conformation capture (Hi-C) technology. The assembled genome was 798.38 Mb with a contig N50 of 1.43 Mb and a scaffold N50 of 32.42 Mb, which were anchored onto 24 pseudochromosomes. A total of 22,019 genes were functionally annotated, which accounted for 95.27% of the predicted protein-coding genes. Chromosomal collinearity analysis revealed chromosome fusion or fission events in Clupeiformes species. Three genetic groups of S. tenuifilis were revealed along the Chinese coast using restriction site-associated DNA sequencing (RADseq). We investigated the influence of four bioclimatic variables as potential drivers of adaptive divergence in S. tenuifilis, suggesting that these environmental variables, especially sea surface temperature, may play important roles as drivers of spatially varying selection for S. tenuifilis. We also identified candidate functional genes underlying adaptive mechanisms and ecological tradeoffs using redundancy analysis (RDA) and BayeScan analysis. In summary, this study sheds light on the evolution and spatial patterns of genetic variation of S. tenuifilis, providing a valuable genomic resource for further biological and genetic studies on this species and other closely related Clupeiformes.

Geometric Remodeling of Nitrilase Active Pocket Based on ALF-Scanning Strategy To Enhance Aromatic Nitrile Substrate Preference and Catalytic Efficiency.

Nitrilase can catalyze nitrile compounds to generate corresponding carboxylic acids. Nitrilases as promiscuous enzymes can catalyze a variety of nitrile substrates, such as aliphatic nitriles, aromatic nitriles, etc. However, researchers tend to prefer enzymes with high substrate specificity and high catalytic efficiency. In this study, we developed an active pocket remodeling (ALF-scanning) based on modulating the geometry of the nitrilase active pocket to alter substrate preference and improve catalytic efficiency. Using this strategy, combined with site-directed saturation mutagenesis, we successfully obtained 4 mutants with strong aromatic nitrile preference and high catalytic activity, W170G, V198L, M197F, and F202M, respectively. To explore the synergistic relationship of these 4 mutations, we constructed 6 double-combination mutants and 4 triple-combination mutants. By combining mutations, we obtained the synergistically enhanced mutant V198L/W170G, which has a significant preference for aromatic nitrile substrates. Compared with the wild type, its specific activities for 4 aromatic nitrile substrates are increased to 11.10-, 12.10-, 26.25-, and 2.55-fold, respectively. By mechanistic dissection, we found that V198L/W170G introduced a stronger substrate-residue π-alkyl interaction in the active pocket and obtained a larger substrate cavity (225.66 Å3 to 307.58 Å3), making aromatic nitrile substrates more accessible to be catalyzed by the active center. Finally, we conducted experiments to rationally design the substrate preference of 3 other nitrilases based on the substrate preference mechanism and also obtained the corresponding aromatic nitrile substrate preference mutants of these three nitrilases and these mutants with greatly improved catalytic efficiency. Notably, the substrate range of SmNit is widened. IMPORTANCE In this study, the active pocket was largely remodeled based on the ALF-scanning strategy we developed. It is believed that ALF-scanning not only could be employed for substrate preference modification but might also play a role in protein engineering of other enzymatic properties, such as substrate region selectivity and substrate spectrum. In addition, the mechanism of aromatic nitrile substrate adaptation we found is widely applicable to other nitrilases in nature. To a large extent, it could provide a theoretical basis for the rational design of other industrial enzymes.

Characterization of Cinnamomum kanehirae Extract-Stimulated Triterpenoids Synthesis in Submerged Fermentation of Antrodia camphorata via Untargeted Metabolomics.

The underlying mechanisms of Cinnamomum kanehirae-stimulated growth and metabolism of Antrodia camphorata remain unknown. Herein, we first observed that the methanol extract of C. kanehirae trunk (MECK) (2 g/L) showed a potent stimulatory effect on A. camphorata triterpenoids production (115.6 mg/L). Second, MECK treatment considerably increased the category and abundance of many secondary metabolites in the mycelia. We identified 93 terpenoids (8 newly formed and 49 upregulated) in the MECK-treated mycelia, wherein 21 terpenoids were the same as those in the fruiting bodies. Third, 42 out of the 93 terpenoids were annotated in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, mainly involving monoterpenoids and diterpenoids syntheses. Finally, 27 monoterpenes and 16 sesquiterpenes were detected in the MECK, and the two terpenoids with the highest abundance (linalool and α-pinene) were selected for verification and found to considerably increase the terpenoids production of A. camphorata and demonstrate the regulation of mRNA expression levels of nine key genes in the mevalonate pathway via RT-qPCR. This study is beneficial for elucidating the terpenoids synthesis mechanism in A. camphorata.

Machine learning based age-authentication assisted by chemo-kinetics: Case study of strong-flavor Chinese Baijiu.

The aged Chinese liquor, Baijiu, is highly valued for its superior organoleptic qualities. However, since age-authentication method and aging-mechanism elucidation of Baijiu is still in the exploratory stage, high-quality aged Baijiu is often replaced by lower-quality, less-aged product with fraudulent mislabeling. Authentic high-quality strong-flavor Baijiu was analyzed by gas chromatography-mass spectrometry. Total esters decreased with aging, while acids, alcohols, aldehydes, ketones, terpenes, pyrazines increased. Although concentrations of partial compounds showed non-monotonic profiling during aging, a close positive linear correlation (R2 = 0.7012) of Baijiu Evenness index (0.55-0.59) with aging time was observed, indicating a more balanced composition in aged Baijiu. The reaction quotient (Qc) of each esterification, calculated by the corresponding reactant and product concentration, approached to the corresponding thermodynamic equilibrium constant Kc. This result demonstrated that the spontaneous transformation driven by thermodynamics explained part of the aging compositional profiling. Furthermore, an aging-related feature selection and an age-authentication method were established based on three models combined with five ranking algorithms. Forty-one key features, including thirty-six compound concentrations, four esterification Qc values and the Evenness index were selected out. The age-authentication based on neural network using forty-one input features accurately predicted the age group of Baijiu samples (F1 = 100 %). These findings have deepened understanding of the Baijiu aging mechanism and provided a novel, effective approach for age-authentication of Baijiu and other liquors.

The differences between broad bean koji fermented in laboratory and factory conditions by an efficient Aspergillus oryzae.

Broad bean paste-meju was fermented by a mixture of broad bean koji and saline; koji fermentation is an essential process for the production of broad bean paste-meju. Aspergillus oryzae was the most widely used in sauce fermentation. The purpose of this study was to research the factory adaptability of the highly efficient A. oryzae PNM003 and further evaluate the effect of fermentation conditions and fermentation strains on koji. A. oryzae PNM003 was compared with the widely used strain HN 3.042 not only in the laboratory but also in factory conditions (large scale). Results showed that the koji made with the same starter in the factory had a greater amount of fungi than that in the laboratory. Bacteria and yeast levels in HN_L koji were higher than in PN_L koji. As for fungi constitution, almost only Aspergillus survived in the end through the microorganism self-purification process during koji fermentation. As for the bacterial constitution, koji was grouped by fermentation conditions instead of fermentation starter. PN koji had higher protease activity and a higher content of total acids, amino acid nitrogen, amino acids, and organic acids in the laboratory conditions. Nevertheless, in factory conditions, PN koji and HN koji had similar indexes. As for volatile flavor compounds, koji made with the two starters in the same condition was grouped together. As for the same starter, there were more flavor compounds metabolized in the factory condition than in the laboratory condition, especially esters and alcohols. The results showed PN was a highly efficient strain to ferment koji, but the advantages were expressed more remarkably in laboratory conditions. In brief, the fermented condition had a greater influence than the fermentation starter for broad bean koji.

Metabolomic analysis of the effects of a mixed culture of Saccharomyces cerevisiae and Lactiplantibacillus plantarum on the physicochemical and quality characteristics of apple cider vinegar.

Introduction: This study compared differences in physicochemical characteristics of the vinegar made by a mixed culture (MC) of Saccharomyces cerevisiae and Lactiplantibacillus plantarum and a pure culture (PC) of Saccharomyces cerevisiae. Methods: The fermentation process was monitored, and metabolomics analysis by Liquid Chromagraphy-Mass Spectrometry (LC-MS) was applied to the compositional differences between PC and MC vinegars, combined with quantification of organic acids, amino acids and B vitamins. Results: A total of 71 differential metabolites including amino acids, organic acids and carbohydrates, and six possible key metabolic pathways were identified. MC enhanced the malic acid utilization and pyruvate acid metabolism during fermentation, increasing substrate-level phosphorylation, and supplying more energy for cellular metabolism. Higher acidity at the beginning of acetic acid fermentation, resulting from lactic acid production by Lactiplantibacillus plantarum in MC, suppressed the cellular metabolism and growth of Acetobacter pasteurianus, but enhanced its alcohol metabolism and acetic acid production in MC. MC vinegar contained more vitamin B, total flavonoids, total organic acids, amino acids and had a higher antioxidant capacity. MC enhanced the volatile substances, particularly ethyl lactate, ethyl caprate and ethyl caproate, which contributed to a stronger fruity aroma. Discussion: These results indicated the mixed culture in alcoholic fermentation can effectively enhance the flavor and quality of apple cider vinegar.