Analysis of the core genome and pangenome of Clostridium butyricum.

Clostridium butyricum is an anaerobic bacterium that inhabits broad niches. Clostridium butyricum is known for its production of butyrate, 1,3-propanediol, and hydrogen. This study aimed to present a comparative pangenome analysis of 24 strains isolated from different niches. We sequenced and annotated the genome of C. butyricum 3-3 isolated from the Chinese baijiu ecosystem. The pangenome of C. butyricum was open. The core genome, accessory genome, and strain-specific genes comprised 1011, 4543, and 1473 genes, respectively. In the core genome, Carbohydrate metabolism was the largest category, and genes in the biosynthetic pathway of butyrate and glycerol metabolism were conserved (in the core or soft-core genome). Furthermore, the 1,3-propanediol operon existed in 20 strains. In the accessory genome, numerous mobile genetic elements belonging to the Replication, recombination, and repair (L) category were identified. In addition, genome islands were identified in all 24 strains, ranging from 2 (strain KNU-L09) to 53 (strain SU1), and phage sequences were found in 17 of the 24 strains. This study provides an important genomic framework that could pave the way for the exploration of C. butyricum and future studies on the genetic diversification of C. butyricum.

The expression, purification, and functional evaluation of the novel tumor suppressor fusion protein IL-24-CN.

Interleukin-24 (IL-24) can specifically induce apoptosis in a broad range of cancer cells without harming normal cells. The interaction of contortrostatin (CN) with integrins on angiogenic vascular endothelial and tumor cells is modulated by the RGD motifs that can significantly inhibit metastasis and angiogenesis. To achieve superior therapeutic efficacy by combining anti-metastasis with tumor-selective apoptosis activity, CN was fused at the C-terminus of IL-24 with a flexible linker (G4S)2, and the recombinant IL-24-CN was expressed in Escherichia coli as a Thioredoxin (Trx)/IL-24-CN fusion protein. The target protein was purified using nickel affinity chromatography. Furthermore, we simplified the purification process by purifying Trx-IL-24-CN and cleaving the Trx tag in one step. The final yield of IL-24-CN was 27.6 mg/L based on flask fermentation. In vitro activity assay demonstrated that the recombinant IL-24-CN could more effectively suppress tumor growth and induce apoptosis of melanoma cells. Scratch and transwell assays suggested that IL-24-CN strongly reduced the migration and invasion behavior of melanoma cells. Immunofluorescence analysis and cell adhesion assay showed that CN could evidently improve the tumor inhibition capability of IL-24 by enhancing the affinity of recombinant protein toward cancer cells. In summary, a highly efficient strategy was developed for producing the bioactive IL-24-CN from prokaryotic cells, supporting IL-24-CN in melanoma therapy.Key points• Efficient heterologous production of recombinant IL-24-CN in E. coli using Trx fusion strategy.• Improved tumor growth suppression and apoptosis induction potency of IL-24-CN.• Enhanced cell adhesion ability of IL-24-CN in cancer cells.

Genome-scale metabolic reconstruction and analysis for Clostridium kluyveri.

Clostridium kluyveri is an anaerobic microorganism that is well-known for producing butyrate and hexanoate using ethanol and acetate. It is also an important bacterium in the production of Chinese strong flavour baijiu (SFB). To obtain a comprehensive understanding of its metabolism, a curated genome-scale metabolic model (GSMM) of C. kluyveri, including 708 genes, 994 reactions, and 804 metabolites, was constructed and named iCKL708. This model was used to simulate the growth of C. kluyveri on different carbon substrates and the results agreed well with the experimental data. The butyrate, pentanoate, and hexanoate biosynthesis pathways were also elucidated. Flux balance analysis indicated that the ratio of ethanol to acetate, as well as the uptake rate of carbon dioxide, affected hexanoate production. The GSMM iCKL708 described here provides a platform to further our understanding and exploration of the metabolic potential of C. kluyveri.

Energy-Saving Electrochemical Hydrogen Production Coupled with Biomass-Derived Isobutanol Upgrading.

The widespread application of electrochemical hydrogen production faces significant challenges, primarily attributed to the high overpotential of the oxygen evolution reaction (OER) in conventional water electrolysis. To address this issue, an effective strategy involves substituting OER with the value-added oxidation of biomass feedstock, reducing the energy requirements for electrochemical hydrogen production while simultaneously upgrading the biomass. Herein, we introduce an electrocatalytic approach for the value-added oxidation of isobutanol, a high energy density bio-fuel, coupled with hydrogen production. This approach offers a sustainable route to produce the valuable fine chemical isobutyric acid under mild condition. The electrodeposited Ni(OH)2 electrocatalyst exhibits exceptional electrocatalytic activity and durability for the electro-oxidation of isobutanol, achieving an impressive faradaic efficiency of up to 92.4 % for isobutyric acid at 1.45 V vs. RHE. Mechanistic insights reveal that side reactions predominantly stem from the oxidative C-C cleavage of isobutyraldehyde intermediate, forming by-products including formic acid and acetone. Furthermore, we demonstrate the electro-oxidation of isobutanol coupled with hydrogen production in a two-electrode undivided cell, notably reducing the electrolysis voltage by approximately 180 mV at 40 mA cm-2. Overall, this work represents a significant step towards improving the cost-effectiveness of hydrogen production and advancing the conversion of bio-fuels.

Revealing microbiota characteristics and predicting flavor-producing sub-communities in Nongxiangxing baijiu pit mud through metagenomic analysis and metabolic modeling.

The microorganisms of the pit mud (PM) of Nongxiangxing baijiu (NXXB) have an important role in the synthesis of flavor substances, and they determine attributes and quality of baijiu. Herein, we utilize metagenomics and genome-scale metabolic models (GSMMs) to investigate the microbial composition, metabolic functions in PM microbiota, as well as to identify microorganisms and communities linked to flavor compounds. Metagenomic data revealed that the most prevalent assembly of bacteria and archaea was Proteiniphilum, Caproicibacterium, Petrimonas, Lactobacillus, Clostridium, Aminobacterium, Syntrophomonas, Methanobacterium, Methanoculleus, and Methanosarcina. The important enzymes ofPMwere in bothGH and GT familymetabolism. A total of 38 high-quality metagenome-assembled genomes (MAGs) were obtained, including those at the family level (n = 13), genus level (n = 17), and species level (n = 8). GSMMs of the 38 MAGs were then constructed. From the GSMMs, individual and community capabilities respectively were predicted to be able to produce 111 metabolites and 598 metabolites. Twenty-three predicted metabolites were consistent with the metabonomics detected flavors and served as targets. Twelve sub-community of were screened by cross-feeding of 38 GSMMs. Of them, Methanobacterium, Sphaerochaeta, Muricomes intestini, Methanobacteriaceae, Synergistaceae, and Caloramator were core microorganisms for targets in each sub-community. Overall, this study of metagenomic and target-community screening could help our understanding of the metabolite-microbiome association and further bioregulation of baijiu.

Optimization of Clostridium beijerinckii semi-solid fermentation of rape straw to produce butyric acid by genome analysis.

Butyric acid is a volatile saturated monocarboxylic acid, which is widely used in the chemical, food, pharmaceutical, energy, and animal feed industries. This study focuses on producing butyric acid from pre-treated rape straw using simultaneous enzymatic hydrolysis semi-solid fermentation (SEHSF). Clostridium beijerinckii BRM001 screened from pit mud of Chinese nongxiangxing baijiu was used. The genome of C. beijerinckii BRM001 was sequenced and annotated. Using rape straw as the sole carbon source, fermentation optimization was carried out based on the genomic analysis of BRM001. The optimized butyric acid yield was as high as 13.86 ± 0.77 g/L, which was 2.1 times higher than that of the initial screening. Furthermore, under optimal conditions, non-sterile SEHSF was carried out, and the yield of butyric acid was 13.42 ± 0.83 g/L in a 2.5-L fermentor. This study provides a new approach for butyric acid production which eliminates the need for detoxification of straw hydrolysate and makes full use of the value of fermentation waste residue without secondary pollution, making the whole process greener and more economical, which has a certain industrial potential.

Genome Analysis and Optimization of Caproic Acid Production of Clostridium butyricum GD1-1 Isolated from the Pit Mud of Nongxiangxing Baijiu.

Caproic acid is a precursor substance for the synthesis of ethyl caproate, the main flavor substance of nongxiangxing baijiu liquor. In this study, Clostridium butyricum GD1-1, a strain with high caproic acid concentration (3.86 g/l), was isolated from the storage pit mud of nongxiangxing baijiu for sequencing and analysis. The strain's genome was 3,840,048 bp in length with 4,050 open reading frames. In addition, virulence factor annotation analysis showed C. butyricum GD1-1 to be safe at the genetic level. However, the annotation results using the Kyoto Encyclopedia of Genes and Genomes Automatic Annotation Server predicted a deficiency in the strain's synthesis of alanine, methionine, and biotin. These results were confirmed by essential nutrient factor validation experiments. Furthermore, the optimized medium conditions for caproic acid concentration by strain GD1-1 were (g/l): glucose 30, NaCl 5, yeast extract 10, peptone 10, beef paste 10, sodium acetate 11, L-cysteine 0.6, biotin 0.004, starch 2, and 2.0% ethanol. The optimized fermentation conditions for caproic acid production by C. butyricum GD1-1 on a single-factor basis were: 5% inoculum volume, 35°C, pH 7, and 90% loading volume. Under optimal conditions, the caproic acid concentration of strain GD1-1 reached 5.42 g/l, which was 1.40 times higher than the initial concentration. C. butyricum GD1-1 could be further used in caproic acid production, NXXB pit mud strengthening and maintenance, and artificial pit mud preparation.

Isolation, Identification, and Fermentation Medium Optimization of a Caproic Acid­Producing Enterococcus casseliflavus Strain from Pit Mud of Chinese Strong Flavor Baijiu Ecosystem.

Caproic acid is the precursor material of ethyl hexanoate, a representative flavor substance in strong flavor baijiu (SFB). Increasing the content of caproic acid in SFB helps to improve its quality. In the present study, caproic acid-producing bacteria from the pit mud of an SFB ecosystem were isolated, purified, and characterized. Strain BF-1 with the highest caproic acid yield (0.88 g/l) was selected. The morphological and molecular identification analysis showed that strain BF-1 was Enterococcus casseliflavus. The genome of E. casseliflavus BF-1 was sequenced and was found to be 2,968,377 bp in length with 3,270 open reading frames (ORFs). The caproic acid biosynthesis pathway in E. casseliflavus BF-1 was predicted based on the KAAS annotation. The virulence factors in the genome of strain BF-1 were annotated, which showed that E. casseliflavus BF-1 is safe at the genetic level. After adding essential nutrients based on the KAAS annotation, the optimum medium conditions for acid production by strain BF-1 were obtained by performing orthogonal experiments. The caproic acid yield of strain BF-1 reached 3.03 g/l, which was 3.44-fold higher than the initial yield. The optimized fer- mentation of caproic acid production by BF-1 was reported for the first time. The strain could be further used to regulate the ecosystem in baijiu production to improve its quality.

Comparative genome analysis of Clostridium beijerinckii strains isolated from pit mud of Chinese strong flavor baijiu ecosystem.

Clostridium beijerinckii is a well-known anaerobic solventogenic bacterium which inhabits a wide range of different niches. Previously, we isolated five butyrate-producing C. beijerinckii strains from pit mud (PM) of strong-flavor baijiu (SFB) ecosystems. Genome annotation of the five strains showed that they could assimilate various carbon sources as well as ammonium to produce acetate, butyrate, lactate, hydrogen, and esters but did not produce the undesirable flavors isopropanol and acetone, making them useful for further exploration in SFB production. Our analysis of the genomes of an additional 233 C. beijerinckii strains revealed an open pangenome based on current sampling and will likely change with additional genomes. The core genome, accessory genome, and strain-specific genes comprised 1567, 8851, and 2154 genes, respectively. A total of 298 genes were found only in the five C. beijerinckii strains from PM, among which only 77 genes were assigned to Clusters of Orthologous Genes categories. In addition, 15 transposase and 12 phage integrase families were found in all five C. beijerinckii strains from PM. Between 18 and 21 genome islands were predicted for the five C. beijerinckii genomes. The existence of a large number of mobile genetic elements indicated that the genomes of the five C. beijerinckii strains evolved with the loss or insertion of DNA fragments in the PM of SFB ecosystems. This study presents a genomic framework of C. beijerinckii strains from PM that could be used for genetic diversification studies and further exploration of these strains.

Research advances on sake rice, koji, and sake yeast: A review.

Sake is the national alcoholic beverage of Japan, and its history can be traced back more than 1300 years. With the development and maturity of the sake-brewing technique, a unique flavor and taste gradually formed, which led to its wide use in Japan and internationally. This paper reviews and discusses the research advances of sake rice, koji, and sake yeast. The various enzymes and involved genes of microbes in the rice koji, and the separation/breeding of sake yeasts are expounded particularly. Moreover, the fields where further research is required are presented. Therefore, this review presents recent comprehensive research details of sake's ingredients and the involved study perspectives.

Advances and Applications of Clostridium Co-culture Systems in Biotechnology.

Clostridium spp. are important microorganisms that can degrade complex biomasses such as lignocellulose, which is a widespread and renewable natural resource. Co-culturing Clostridium spp. and other microorganisms is considered to be a promising strategy for utilizing renewable feed stocks and has been widely used in biotechnology to produce bio-fuels and bio-solvents. In this review, we summarize recent progress on the Clostridium co-culture system, including system unique advantages, composition, products, and interaction mechanisms. In addition, biochemical regulation and genetic modifications used to improve the Clostridium co-culture system are also summarized. Finally, future prospects for Clostridium co-culture systems are discussed in light of recent progress, challenges, and trends.

Effects of Saccharomycopsis fibuligera and Saccharomyces cerevisiae inoculation on small fermentation starters in Sichuan-style Xiaoqu liquor.

Xiaoqu liquor is a type of distilled spirit in China prepared on a small scale from a small solid starter culture. Although this liquor is popular in southwestern China, it can have a dull taste, limiting its market. To improve the flavour profile of Xiaoqu liquor, we selected two functional yeast strains (Saccharomycopsis fibuligera and Saccharomyces cerevisiae) from Zaopei (fermented grain) of Baijiu liquor and used them for Xiaoqu liquor fermentation. Compared with traditional Xiaoqu (Starter), bioaugmentation inoculation increased the glucoamylase and acidic protease activities and the ethanol synthesis rate, while decreasing the acidity of the Zaopei (fermented grains) in the early stage of fermentation. By the end of the fermentation process, the alcohol and ester content had also increased by 42.5% and 11.8%, respective, and that of aldehydes and ketones, and heterocyclic compounds decreased by 73.7% and 77.1%, respectively. Traditional isolation and high-throughput sequencing were employed to analyse the microorganisms in the Zaopei. Bioaugmentation inoculation increased the microbial diversity of Xiaoqu liquor during the fermentation process. The dominant fungus during fermentation using the two types of starter cultures was S. cerevisiae, whereas the dominant bacteria was Pseudomonas, followed by Bacillus, Weissella, Lactobacillus, and Bacteroides. Principal component analysis of the bacterial community structure and flavour substances in the Zaopei produced using the two strains revealed that there were few differences between the two liquors and that inoculation with functional yeasts may not change the flavour substances in Xiaoqu liquor. However, correlation analysis showed that Escherichia Shigella, Terrisporobacter, Bacillus, Clostridium, and Prevotellaceae are the main microorganisms in the Xiaoqu liquor fermentation process. These results lay the foundation to improve the quality of Xiaoqu liquor.

Network pharmacology suggests biochemical rationale for treating COVID-19 symptoms with a Traditional Chinese Medicine.

Chinese herbal formulas including the lung-cleaning and toxicity-excluding (LCTE) soup have played an important role in treating the ongoing COVID-19 pandemic (caused by SARS-CoV-2) in China. Applying LCTE outside of China may prove challenging due to the unfamiliar rationale behind its application in terms of Traditional Chinese Medicine. To overcome this barrier, a biochemical understanding of the clinical effects of LCTE is needed. Here, we explore the chemical compounds present in the reported LCTE ingredients and the proteins targeted by these compounds via a network pharmacology analysis. Our results indicate that LCTE contains compounds with the potential to directly inhibit SARS-CoV-2 and inflammation, and that the compound targets proteins highly related to COVID-19's main symptoms. We predict the general effect of LCTE is to affect the pathways involved in viral and other microbial infections, inflammation/cytokine response, and lung diseases. Our work provides a biochemical basis for using LCTE to treat COVID-19 and its main symptoms.

Wheat Qu and Its Production Technology, Microbiota, Flavor, and Metabolites.

Chinese rice wine (CRW) is an ancient wine with a moderate alcohol content; it has been prized in China for its color, sweet flavor profile, and is a national beverage typically used in banquets. Wheat Qu is the starter used to make CRW, and plays a vital role in the quality and flavor profile. This review details the raw materials and the wheat Qu manufacturing process, and recent information on wheat Qu microbiotas. The volatile compounds in wheat Qus and its flavor are also reviewed. Finally, the enzymes, and metabolites contained in wheat Qu, such as biogenic amines, are also discussed.

Research Progress on Flavor Compounds and Microorganisms of Maotai Flavor Baijiu.

Maotai-flavor liquor is one of the three basic traditional Chinese baijiu and is also the most famous baijiu in the world. Guizhou Maotai baijiu is the representative of Maotai-flavor liquor, which has a long history of culture and is prepared using unique brewing methods. However, the main flavor of Maotai-flavor liquor as well as the mechanism by which its aroma is produced is unclear. In this review, the Da-qu production and fermentation processes for Maotai-flavor liquor are briefly described along with the flavoring constituents of Maotai-flavor liquor that have been recently reported. In addition, the volatile compounds and the aroma derived from Maotai-flavor liquor are discussed. Finally, the microorganisms for the high-temperature Daqu and fermentation processes of Maotai-flavor liquor are discussed. PRACTICAL APPLICATION: Maotai is one of the most famous baijiu in China and the most valuable in the market. However, it is unclear what is the key flavor of Maotai and what microbial metabolism is produced. So, if we can figure out the key flavor substances of Maotai baijiu, we can use the various technology to explore the microbes that produce this flavor to understand the mechanism of the production of Maotai. This will not only achieve breakthroughs in academic value, but also bring higher value to Maotai. On this basis, we can brew Maotai baijiu with better quality according to the fermentation mechanism of Maotai.

Assessment of ß-glucans, phenols, flavor and volatile profiles of hulless barley wine originating from highland areas of China.

Low alcohol hulless barley wine (HW) is a popular beverage among the highland areas in China. It is known to have several health benefits due to the presence of ß-glucan and antioxidant compounds. Therefore, the total ß-glucan content, total phenols and flavonoids of HW samples from the highland areas of Sichuan province and Tibet were determined in this study. The results indicated that HW is abundant in both ß-glucan (54-76 mg/L) and phenolic compounds (131-178 mg/L). Moreover, this study also investigated the flavor and aroma characteristics of HW samples. A total of forty six volatile aroma substances were identified by GC-MS. The HWs could be classified into three distinct groups in terms of the region of origin according to the results of PCA based on the GC-MS data. These findings provide a useful foundation for further study of the health benefits and the flavor characteristics of HW in highland areas.

Diversity, Function, and Application of Clostridium in Chinese Strong Flavor Baijiu Ecosystem: A Review.

Baijiu is a Chinese traditional distilled liquor with an annual yield over 13.12 million tons. Strong flavor baijiu (SFB) also called Luzhou-flavor liquor, takes account for > 70% of the total baijiu produced. SFB is produced by an open solid fermentation process with a complex microbial ecosystem. Clostridium is one of the most important microorganisms for the formation of the main flavor compounds of SFB, such as ethyl caproate. In this paper, we review current research progress on the Clostridium in the SFB ecosystem, focusing on the species diversity, physiological and metabolic features along with interspecies interactions. Systems biology approaches for the study of Clostridium from SFB ecosystems were discussed and explored. Furthermore, current applications of Clostridium in SFB production were discussed. PRACTICAL APPLICATION: Strong flavor baijiu (SFB) accounts for more than 70% of total yield of Chinese baijiu, which exists for hundreds of years. Clostridium is common in SFB ecosystem and identified to be one of main contributors of flavor compounds in SFB. Study on the Clostridium from SFB ecosystem is not only helpful for the understanding of flavor compounds formation mechanism, but also the improvement of SFB quality. This study focuses on the current researches on the Clostridium species in SFB ecosystem, including the species diversity, physiological and metabolic features, and applications.

Reconstruction and analysis of a genome-scale metabolic model of Methylovorus sp. MP688, a high-level pyrroloquinolone quinone producer.

Methylovorus sp. MP688 is a methylotrophic bacterium that can be used as a pyrroloquinolone quinone (PQQ) producer. To obtain a comprehensive understanding of its metabolic capabilities, we constructed a genome-scale metabolic model (iWZ583) of Methylovorus sp. MP688, based on its genome annotations, data from public metabolic databases, and literature mining. The model includes 772 reactions, 764 metabolites, and 583 genes. Growth of Methylovorus sp. MP688 was simulated using different carbon and nitrogen sources, and the results were consistent with experimental data. A core metabolic essential gene set of 218 genes was predicted by gene essentiality analysis on minimal medium containing methanol. Based on in silico predictions, the addition of aspartate to the medium increased PQQ production by 4.6- fold. Deletion of three reactions associated with four genes (MPQ_1150, MPQ_1560, MPQ_1561, MPQ_1562) was predicted to yield a PQQ production rate of 0.123 mmol/gDW/h, while cell growth decreased by 2.5%. Here, model iWZ583 represents a useful platform for understanding the phenotype of Methylovorus sp. MP688 and improving PQQ production.