Semiautomated design and soluble expression of a chimeric antigen TbpAB01 from Glaesserella parasuis.

Pathogenic bacterial membrane proteins (MPs) are a class of vaccine and antibiotic development targets with widespread clinical application. However, the inherent hydrophobicity of MPs poses a challenge to fold correctly in living cells. Herein, we present a comprehensive method to improve the soluble form of MP antigen by rationally designing multi-epitope chimeric antigen (ChA) and screening two classes of protein-assisting folding element. The study uses a homologous protein antigen as a functional scaffold to generate a ChA possessing four epitopes from transferrin-binding protein A of Glaesserella parasuis. Our engineered strain, which co-expresses P17 tagged-ChA and endogenous chaperones groEL-ES, yields a 0.346 g/L highly soluble ChA with the property of HPS-positive serum reaction. Moreover, the protein titer of ChA reaches 4.27 g/L with >90% soluble proportion in 5-L bioreactor, which is the highest titer reported so far. The results highlight a timely approach to design and improve the soluble expression of MP antigen in industrially viable applications.

Engineering protein translocation and unfolded protein response enhanced human PH-20 secretion in Pichia pastoris.

Hyaluronidases catalyze the degradation of hyaluronan (HA), which is finding rising applications in medicine, cosmetic, and food industries. Recombinant expression of hyaluronidases in microbial hosts has been given special attention as a sustainable way to substitute animal tissue-derived hyaluronidases. In this study, we focused on optimizing the secretion of hyaluronidase from Homo sapiens in Pichia pastoris by secretion pathway engineering. The recombinant hyaluronidase was first expressed under the control of a constitutive promoter PGCW14. Then, two endoplasmic reticulum-related secretory pathways were engineered to improve the secretion capability of the recombinant strain. Signal peptide optimization suggested redirecting the protein into co-translational translocation using the ost1-proα signal sequence improved the secretion level by 20%. Enhancing the co-translational translocation by overexpressing signal recognition particle components further enhanced the secretory capability by 48%. Then, activating the unfolded protein response by overexpressing a transcriptional factor ScHac1p led to a secreted hyaluronidase activity of 4.06 U/mL, which was 2.1-fold higher than the original strain. Finally, fed-batch fermentation elevated the production to 19.82 U/mL. The combined engineering strategy described here could be applied to enhance the secretion capability of other proteins in yeast hosts. KEY POINTS: • Improving protein secretion by enhancing co-translational translocation in P. pastoris was reported for the first time. • Overexpressing Hac1p homologous from different origins improved the rhPH-20 secretion. • A 4.9-fold increase in rhPH-20 secretion was achieved after fermentation optimization and fed-batch fermentation.

Improving the soluble expression of difficult-to-express proteins in prokaryotic expression system via protein engineering and synthetic biology strategies.

Solubility and folding stability are key concerns for difficult-to-express proteins (DEPs) restricted by amino acid sequences and superarchitecture, resolved by the precise distribution of amino acids and molecular interactions as well as the assistance of the expression system. Therefore, an increasing number of tools are available to achieve efficient expression of DEPs, including directed evolution, solubilization partners, chaperones, and affluent expression hosts, among others. Furthermore, genome editing tools, such as transposons and CRISPR Cas9/dCas9, have been developed and expanded to construct engineered expression hosts capable of efficient expression ability of soluble proteins. Accounting for the accumulated knowledge of the pivotal factors in the solubility and folding stability of proteins, this review focuses on advanced technologies and tools of protein engineering, protein quality control systems, and the redesign of expression platforms in prokaryotic expression systems, as well as advances of the cell-free expression technologies for membrane proteins production.

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.

Heterologous expression, fermentation strategies and molecular modification of collagen for versatile applications.

Collagen is a kind of high macromolecular protein with unique tissue distribution and distinctive functions in the body. At present, most collagen products are extracted from the tissues and organs of mammals or marine fish. However, this method exhibits several disadvantages, including low efficiency and serious waste generation, which makes it difficult to meet the current market demand. With the rapid development of synthetic biology and the deepening of high-density fermentation technology, the collagen preparation by biosynthesis strategy emerges as the times require. Co-expression with the proline hydroxylase gene can solve the problem of non-hydroxylated collagen, but the yield may be affected. Therefore, improving the expression through molecular modification and dynamic regulation of synthesis is an entry point for future research. Due to the defects in certain properties of the natural collagen, modification of properties would be benefit for meeting the requirements of practical application. In this paper, in-depth investigations on recombinant expression, fermentation, and modification studies of collagen are conducted. Also, it summarizes the research progress of collagen in food, medicine, and beauty industry in recent years. Furthermore, the future development trend and application prospect of collagen are discussed, which would provide guidance for its preparation and application.

Technology and functional insights into the nicotinamide mononucleotide for human health.

Nicotinamide mononucleotide (NMN), a naturally occurring biologically active nucleotide, mainly functions via mediating the biosynthesis of NAD+. In recent years, its excellent pharmacological activities including anti-aging, treating neurodegenerative diseases, and protecting the heart have attracted increasing attention from scholars and entrepreneurs for production of a wide range of formulations, including functional food ingredients, health care products, active pharmaceuticals, and pharmaceutical intermediates. Presently, the synthesis methods of NMN mainly include two categories: chemical synthesis and biosynthesis. With the development of biocatalyst engineering and synthetic biology strategies, bio-preparation has proven to be efficient, economical, and sustainable methods. This review summarizes the chemical synthesis and biosynthetic pathways of NMN and provides an in-depth investigation on the mining and modification of enzyme resources during NMN biosynthesis, as well as the screening of hosts and optimization of chassis cells via metabolic engineering, which provide effective strategies for efficient production of NMN. In addition, an overview of the significant physiological functions and activities of NMN is elaborated. Finally, future research on technical approaches to further enhance NMN synthesis and strengthen clinical studies of NMN are prospected, which would lay the foundation for further promoting the application of NMN in nutrition, healthy food, and medicine in the future. KEY POINTS: • NMN supplementation effectively increases the level of NAD+. • The chemical and biological synthesis of NMN are comprehensively reviewed. • The impact of NMN on the treatment of various diseases is summarized.

Constructing a Defined Starter for Multispecies Vinegar Fermentation via Evaluation of the Vitality and Dominance of Functional Microbes in an Autochthonous Starter.

Mature vinegar culture has usually been used as a type of autochthonous starter to rapidly initiate the next batch of acetic acid fermentation (AAF) and maintain the batch-to-batch uniformity of AAF in the production of traditional cereal vinegar. However, the vitality and dominance of functional microbes in autochthonous starters remain unclear, which hinders further improvement of fermentation yield and production. Here, based on metagenomic (MG), metatranscriptomic (MT), and 16S rRNA gene sequencings, 11 bacterial operational taxonomic units (OTUs) with significant metabolic activity (MT/MG ratio >1) and dominance (relative abundance >1%) were targeted in the autochthonous vinegar starter, all of which were assigned to 4 species (Acetobacter pasteurianus, Lactobacillus acetotolerans, L. helveticus, Acetilactobacillus jinshanensis). Then, we evaluated the successions and interactions of these 11 bacterial OTUs at different AAF stages. Last, a defined starter was constructed with 4 core species isolated from the autochthonous starter (A. pasteurianus, L. acetotolerans, L. helveticus, Ac. jinshanensis). The defined starter culture could rapidly initiate the AAF in a sterile or unsterilized environment, and similar dynamics of metabolites (ethanol, titratable acidity, acetic acid, lactic acid, and volatile compounds) and environmental indexes (temperature, pH) of fermentation were observed as compared with that of autochthonous starter (P > 0.05). This work provides a method to construct a defined microbiota from a complex system while preserving its metabolic function. IMPORTANCE Complex microorganisms are beneficial to the flavor formation in natural food fermentation, but they also pose challenges to the mass production of standardized products. It is attractive to construct a defined starter to rapidly initiate fermentation process and significantly improve fermentation yield. This study provides a comprehensive understanding of vital and dominant species in the autochthonous vinegar starter via multi-omics, and designs a defined microbial community for the efficient fermentation of cereal vinegar.

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.

Preparation and applications of keratin biomaterials from natural keratin wastes.

Keratin is a kind of natural polymer that is abundant in feathers, wool, and hair. Being one of the natural biomolecules, keratin has excellent biological activity, biocompatibility, biodegradability, favorable material mechanical properties, and natural abundance, which exhibit significant biological and biomedical application potentials. At present, the strategies commonly used for preparing keratin from hair, feathers, wool, etc. include physical, chemical, and enzymatic methods. The present article mainly reviews the structure, classification, preparation methods, and the main biological applications of keratin, and these applications cover wound healing, hemostasis, targeted release of tissue engineering drugs, and so on. It is expected to lay the foundations for its future in-depth investigations and wide applications of keratin biomaterials. KEY POINTS: • There are several pathways to prepare biologically active keratin from wool, feathers, and human hair, etc • Promoting blood coagulation by keratin is related to the adhesion and activation of platelets and the aggregation of fibrin • The biological applications of keratin, including wound healing and tissue engineering, are summarized.

Pathway engineering facilitates efficient protein expression in Pichia pastoris.

Pichia pastoris has been recognized as an important platform for the production of various heterologous proteins in recent years. The strong promoter AOX1, induced by methanol, with the help of the α-pre-pro signal sequence, can lead to a high expression level of extracellular protein. However, this combination was not always efficient, as protein secretion in P. pastoris involves numerous procedures mediated by several cellular proteins, including folding assisted by endoplasmic reticulum (ER) molecular chaperones, degradation through ubiquitination, and an efficient vesicular transport system. Efficient protein expression requires the cooperation of various intracellular pathways. This article summarizes the process of protein secretion, modification, and transportation in P. pastoris. In addition, the roles played by the key proteins in these processes and the corresponding co-expression effects are also listed. It is expected to lay the foundation for the industrial protein production of P. pastoris. KEY POINTS: • Mechanisms of chaperones in protein folding and their co-expression effects are summarized. • Protein glycosylation modifications are comprehensively reviewed. • Current dilemmas in the overall protein secretion pathway of Pichia pastoris and corresponding solutions are demonstrated.

Effectiveness and safety of KunXian capsule for the treatment of IgA nephropathy.

BACKGROUND: Tripterygium Wilfordii Hook F (TwHF) preparation has been widely used in the treatments of IgA nephropathy (IgAN) in China. However, the effectiveness and safety of the new generation of TwHF preparation, KuxXian capsule, on the treatment of IgAN remains unknown. METHODS: Here, we retrospectively describe our experience treating 55 consecutive IgAN patients with KunXian. We defined complete remission as proteinuria < 0.5 g/24 h and partial remission as proteinuria < 1 g/24 h, each also having > 50% reduction in proteinuria from baseline. RESULTS: At first follow-up after KunXian treatment (5.7 weeks, IQR 4.7-7.9), all but two patients (96%) showed a reduction in proteinuria. The overall median proteinuria decreased from 2.23 g/day at baseline to 0.94 g/day (P < 0.001) at the first follow-up. During a median follow-up of 28 weeks after KunXian administration, 25(45.5%) patients achieved complete remission, 34 (61.8%) patients achieved complete/partial remission. Of the 12 patients discontinued KunXian treatment during the follow-up, the median proteinuria was increased from 0.97 g/24 h to 2.74 g/24 h after a median of 10.9 weeks (P = 0.004). Multivariable Cox models showed that female, treatment switching from previous generation of TwHF preparation, lower initial KunXian dosage, and higher proteinuria at baseline were independently associated proteinuria remission. Of the 20 pre-menopausal females, 12 of them developed oligomenorrhea or menstrual irregularity and ten of them developed amenorrhea. CONCLUSION: KunXian is effectiveness and safety for the treatment of IgA nephropathy. Woman of childbearing age to be informed of the risk of ovarian failure after being treated with TwHF preparations.

Mining the Factors Driving the Evolution of the Pit Mud Microbiome under the Impact of Long-Term Production of Strong-Flavor Baijiu.

The mud cellar creates a unique microenvironment for the fermentation of strong-flavor baijiu (SFB). Recent research and long-term practice have highlighted the key roles of microbes inhabiting pit mud in the formation of SFB's characteristic flavor. A positive correlation between the quality of SFB and cellar age was extracted from practice; however, the evolutionary patterns of pit mud microbiome and driving factors remain unclear. Here, based on the variation regularity analysis of microbial community structure and metabolites of samples from cellars of different ages (∼30/100/300 years), we further investigated the effects of lactate and acetate (main microbial metabolites in fermented grains) on modulating the pit mud microbiome. Esters (50.3% to 64.5%) dominated the volatile compounds identified in pit mud, and contents of the four typical acids (lactate, hexanoate, acetate, and butyrate) increased with cellar age. Bacteria (9.5 to 10.4 log10 [lg] copies/g) and archaea (8.3 to 9.1 lg copies/g) mainly constituted pit mud microbiota, respectively dominated by Clostridia (39.7% to 81.2%) and Methanomicrobia (32.8% to 92.9%). An upward trend with cellar age characterized the relative and absolute abundance of the most predominant bacterial and archaeal genera, Caproiciproducens and Methanosarcina. Correlation analysis revealed significantly (P < 0.05) positive relationships between the two genera and major metabolites. Anaerobic fermentation with acetate and lactate as carbon sources enhanced the enrichment of Clostridia, and furthermore, the relative abundance of Caproiciproducens (40.9%) significantly increased after 15-day fed-batch fermentation with lactate compared with the initial pit mud (0.22%). This work presents a directional evolutionary pattern of pit mud microbial consortia and provides an alternative way to accelerate the enrichment of functional microbes. IMPORTANCE The solid-state anaerobic fermentation in a mud cellar is the most typical feature of strong-flavor baijiu (SFB). Metabolites produced by microbes inhabiting pit mud are crucial to create the unique flavor of SFB. Accordingly, craftspeople have always highlighted the importance of the pit mud microbiome and concluded by centuries of practice that the production rate of high-quality baijiu increases with cellar age. To deepen the understanding of the pit mud microbiome, we determined the microbial community and metabolites of different-aged pit mud, inferred the main functional groups, and explored the forces driving the microbial community evolution through metagenomic, metabolomic, and multivariate statistical analyses. The results showed that the microbial consortia of pit mud presented a regular and directional evolutionary pattern under the impact of continuous batch-to-batch brewing activities. This work provides insight into the key roles of the pit mud microbiome in SFB production and supports the production optimization of high-quality pit mud.

Phospholipids (PLs) know-how: exploring and exploiting phospholipase D for its industrial dissemination.

Owing to their numerous nutritional and bioactive functions, phospholipids (PLs), which are major components of biological membranes in all living organisms, have been widely applied as nutraceuticals, food supplements, and cosmetic ingredients. To date, PLs are extracted solely from soybean or egg yolk, despite the diverse market demands and high cost, owing to a tedious and inefficient manufacturing process. A microbial-based manufacturing process, specifically phospholipase D (PLD)-based biocatalysis and biotransformation process for PLs, has the potential to address several challenges associated with the soybean- or egg yolk-based supply chain. However, poor enzyme properties and inefficient microbial expression systems for PLD limit their wide industrial dissemination. Therefore, sourcing new enzyme variants with improved properties and developing advanced PLD expression systems are important. In the present review, we systematically summarize recent achievements and trends in the discovery, their structural properties, catalytic mechanisms, expression strategies for enhancing PLD production, and its multiple applications in the context of PLs. This review is expected to assist researchers to understand current advances in this field and provide insights for further molecular engineering efforts toward PLD-mediated bioprocessing.

Novisyntrophococcus fermenticellae gen. nov., sp. nov., isolated from an anaerobic fermentation cellar of Chinese strong-flavour baijiu.

A Gram-stain-negative, coccus-shaped, obligately anaerobic, non-motile and non-spore-forming bacterium, designated strain JN500902T, was isolated from the mud in a fermentation cellar used continuously over 30 years for Chinese strong-flavour baijiu production. Colonies were white, circular, convex and smooth-edged. Growth was observed at 20-40 °C (optimum, 37 °C), at pH 5.0-10 (optimum, pH 7.5), with 0-2 % (w/v) NaCl and with 0-4 % (v/v) ethanol. The Biolog assay demonstrated positive reactions of strain JN500902T in the metabolism of l-fucose and pyruvate. The predominant cellular fatty acids (>10 %) consisted of C16 : 0 and C14 : 0. The major end metabolites of strain JN500902T were acetic acid and ethanol when incubated anaerobically in liquid reinforced clostridial medium. Acetate was the major organic acid end product. The complete genome size of strain JN500902T was 3 420 321 bp with 3327 identified genes. The G+C content was 43.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences affiliated strain JN500902T with the family Lachnospiraceae, having low sequence similarity (92.8 %) to the nearest type strain, Syntrophococcus sucromutans DSM 3224T and forming a clearly distinct branch. Core genome phylogenetic analysis of the isolate and 134 strains belonging to the family Lachnospiraceae also revealed that strain JN500902T was well-separated from other genera of this family as a monophyletic clade. The average nucleotide identity and amino acid identity values between strain JN500902T and 134 Lachnospiraceae strains were less than 74 and 65 %, respectively. Considering its polyphasic characteristics, strain JN500902T represents a novel genus and species within the family Lachnospiraceae, for which the name Novisyntrophococcus fermenticellae gen. nov., sp. nov. is proposed. The type strain is JN500902T (=CICC 24502T=JCM 33939T).

Daqu microbiota exhibits species-specific and periodic succession features in Chinese baijiu fermentation process.

Daqu, a brick-shaped product spontaneously fermented under an open environment, has been regarded as the starter of fermentation, raw enzyme preparation and raw materials for baijiu production. However, its contribution in baijiu fermentation has not been fully elaborated yet. Here, the effects of daqu microbiota on baijiu fermentation were investigated under both field-scale and lab-scale conditions. In field-scale baijiu fermentation, the dominant daqu microbes (average relative abundance>10.0%), including unclassified_Leuconostocaceae, Thermoascus, and Thermomyces, tended to dominate the early stage (0-7 d). However, the rare daqu microbes (average relative abundance <0.1%, e.g., Kazachstania) tended to dominate the middle and late stages (11-40 d). In addition, some genera showed differences in species diversity between daqu and fermented grains. The average relative abundance of Lactobacillus was over 75% during baijiu fermentation, and most of them were affiliated with Lactobacillus acetotolerans, while Lactobacillus crustorum dominated the Lactobacillus OTUs in daqu. The similar patterns were also observed during lab-scale baijiu fermentation. The results of function prediction showed the enriched metabolic pathways were associated with glycolysis and long-chain fatty acid esters in baijiu fermentation. These results improved the understanding of daqu microbiota function during baijiu fermentation and provided a basic theory to support the regulation of baijiu production.

A Bottom-Up Approach To Develop a Synthetic Microbial Community Model: Application for Efficient Reduced-Salt Broad Bean Paste Fermentation.

Humans have used high salinity for the production of bean-based fermented foods over thousands of years. Although high salinity can inhibit the growth of harmful microbes and select functional microbiota in an open environment, it also affects fermentation efficiency of bean-based fermented foods and has a negative impact on people's health. Therefore, it is imperative to develop novel defined starter cultures for reduced-salt fermentation in a sterile environment. Here, we explored the microbial assembly and function in the fermentation of traditional Chinese broad bean paste with 12% salinity. The results revealed that the salinity and microbial interactions together drove the dynamic of community and pointed out that five dominant genera (Staphylococcus, Bacillus, Weissella, Aspergillus, and Zygosaccharomyces) may play different key roles in different fermentation stages. Then, core species were isolated from broad bean paste, and their salinity tolerance, interactions, and metabolic characteristics were evaluated. The results provided an opportunity to validate in situ predictions through in vitro dissection of microbial assembly and function. Last, we reconstructed the synthetic microbial community with five strains (Aspergillus oryzae, Bacillus subtilis, Staphylococcus gallinarum, Weissella confusa, and Zygosaccharomyces rouxii) under different salinities and realized efficient fermentation of broad bean paste for 6 weeks in a sterile environment with 6% salinity. In general, this work provided a bottom-up approach for the development of a simplified microbial community model with desired functions to improve the fermentation efficiency of bean-based fermented foods by deconstructing and reconstructing the microbial structure and function.IMPORTANCE Humans have mastered high-salinity fermentation techniques for bean-based fermented product preparation over thousands of years. High salinity was used to select the functional microbiota and conducted food fermentation production with unique flavor. Although a high-salinity environment is beneficial for suppressing harmful microbes in the open fermentation environment, the fermentation efficiency of functional microbes is partially inhibited. Therefore, application of defined starter cultures for reduced-salt fermentation in a sterile environment is an alternative approach to improve the fermentation efficiency of bean-based fermented foods and guide the transformation of traditional industry. However, the assembly and function of self-organized microbiota in an open fermentation environment are still unclear. This study provides a comprehensive understanding of microbial function and the mechanism of community succession in a high-salinity environment during the fermentation of broad bean paste so as to reconstruct the microbial community and realize efficient fermentation of broad bean paste in a sterile environment.

Targeting Follistatin like 1 ameliorates liver fibrosis induced by carbon tetrachloride through TGF-ß1-miR29a in mice.

BACKGROUND: Hepatic fibrosis is a pathological response of the liver to a variety of chronic stimuli. Hepatic stellate cells (HSCs) are the major source of myofibroblasts in the liver. Follistatin like 1 (Fstl1) is a secreted glycoprotein induced by transforming growth factor-ß1 (TGF-ß1). However, the precise functions and regulation mechanisms of Fstl1 in liver fibrogenesis remains unclear. METHODS: Hepatic stellate cell (HSC) line LX-2 stimulated by TGF-ß1, primary culture of mouse HSCs and a model of liver fibrosis induced by CCl4 in mice was used to assess the effect of Fstl1 in vitro and in vivo. RESULTS: Here, we found that Fstl1 was significantly up regulated in human and mouse fibrotic livers, as well as activated HSCs. Haplodeficiency of Fstl1 or blockage of Fstl1 with a neutralizing antibody 22B6 attenuated CCl4-induced liver fibrosis in vivo. Fstl1 modulates TGF-ß1 classic Samd2 and non-classic JNK signaling pathways. Knockdown of Fstl1 in HSCs significantly ameliorated cell activation, cell migration, chemokines C-C Motif Chemokine Ligand 2 (CCL2) and C-X-C Motif Chemokine Ligand 8 (CXCL8) secretion and extracellular matrix (ECM) production, and also modulated microRNA-29a (miR29a) expression. Furthermore, we identified that Fstl1 was a target gene of miR29a. And TGF-ß1 induction of Fstl1 expression was partially through down regulation of miR29a in HSCs. CONCLUSIONS: Our data suggests TGF-ß1-miR29a-Fstl1 regulatory circuit plays a key role in regulation the HSC activation and ECM production, and targeting Fstl1 may be a strategy for the treatment of liver fibrosis. Video Abstract.

Deciphering the d-/l-lactate-producing microbiota and manipulating their accumulation during solid-state fermentation of cereal vinegar.

Symphony orchestra of multi-microorganisms characterizes the solid-state acetic acid fermentation process of Chinese cereal vinegars. Lactate is the predominant non-volatile acid and plays indispensable roles in flavor formation. This study investigated the microbial consortia driving the metabolism of D-/l-lactate during fermentation. Sequencing analysis based on D-/l-lactate dehydrogenase genes demonstrated that Lactobacillus (relative abundance: > 95%) dominated the production of both d-lactate and l-lactate, showing species-specific features between the two types. Lactobacillus helveticus (>65%) and L. reuteri (~80%) respectively dominated l- and d-lactate-producing communities. D-/l-lactate production and utilization capabilities of eight predominant Lactobacillus strains were determined by culture-dependent approach. Subsequently, D-/l-lactate producer L. plantarum M10-1 (d:l ≈ 1:1), l-lactate producer L. casei 21M3-1 (D:L ≈ 0.2:9.8) and D-/l-lactate utilizer Acetobacter pasteurianus G3-2 were selected to modulate the metabolic flux of D-/l-lactate of microbial consortia. The production ratio of D-/l-lactate was correspondingly shifted coupling with microbial consortia changes. Bioaugmentation with L.casei 21M3-1 merely enhanced l-lactate production, displaying ~4-fold elevation at the end of fermentation. Addition of L.plantarum M10-1 twice increased both D- and l-lactate production, while A. pasteurianus G3-2 decreased the content of D-/l-isomer. Our results provided an alternative strategy to specifically manipulate the metabolic flux within microbial consortia of certain ecological niches.

Improving the biocatalytic performance of co-immobilized cells harboring nitrilase via addition of silica and calcium carbonate.

To improve nicotinic acid (NA) yield and meet industrial application requirements of sodium alginate-polyvinyl alcohol (SA-PVA) immobilized cells of Pseudomonas putida mut-D3 harboring nitrilase, inorganic materials were added to the SA-PVA immobilized cells to improve mechanical strength and mass transfer performance. The concentrations of inorganic materials were optimized to be 2.0% silica and 0.6% CaCO3. The optimal pH and temperature for SA-PVA immobilized cells and composite immobilized cells were both 8.0 and 45 °C, respectively. The half-lives of composite immobilized cells were 271.48, 150.92, 92.92 and 33.12 h, which were 1.40-, 1.35-, 1.22- and 1.63-fold compared to SA-PVA immobilized cells, respectively. The storage stability of the composite immobilized cells was slightly increased. The composite immobilized cells could convert 14 batches of 3-cyanopyridine with feeding concentration of 250 mM and accumulate 418 g ·L-1 nicotinic acid, while the SA-PVA immobilized cells accumulated 346 g L-1 nicotinic acid.

Clostridium fermenticellae sp. nov., isolated from the mud in a fermentation cellar for the production of the Chinese liquor, baijiu.

A novel Gram-stain-positive, rod-shaped, obligately anaerobic, non-motile, spore-forming and binary fission encapsulated bacterium, designated strain JN500901T, was isolated from a mud cellar which has been continuously used for the fermentation of Chinese strong-flavour baijiu for over 100 years. Growth of JN500901Toccurred at pH 4.5-8.0 (optimum, pH 5.0), 20-40 °C (37 °C), 0-2 % (w/v) NaCl and 0-10 % (v/v) ethanol. The Biolog assay revealed that strain JN500901T metabolized d-fructose, l-fucose, isomaltulose and l-rhamnose among the 95 studied carbon sources. p-Cresol was the predominant volatile metabolite in the fermentation broth of strain JN500901T incubated in liquid reinforced clostridial medium under anaerobic conditions. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain JN500901T belongs to Clostridiumsensu stricto, and shared the highest sequence similarity to Clostridiumcarboxidivorans DSM 15243T (94.2 %), followed by Clostridiumscatologenes DSM 757T (94.1 %). The dominant cellular fatty acids (>10 %) were C16 : 0 FAME (36.6 %), C19 : 0 cyc 9,10 DMA (19.8 %) and C16 : 1 cis 9 DMA (11.8 %). The complete genome of strain JN500901T contained a circular chromosome of 2.812 Mb with 2611 genes and 31.0 mol% G+C content. Comparative genome analysis of the strain JN500901T, Clostridiumcarboxidivorans DSM 15243T and Clostridiumscatologenes DSM 757T revealed 74.5 and 74.8 % average nucleotide identity, respectively. Based on the phenotypic, biochemical and phylogenetic analyses presented here, strain JN500901T is considered to be a novel species of the genus Clostridiumsensustricto, for which the name Clostridium fermenticellae sp. nov. is proposed. The type strain is JN500901T (=CICC 24501T=JCM 32827T).