Trillions of microbes harbor the gastrointestinal tract and co-exist peacefully with the human host. Microbial diversity and composition among individuals depend upon human age, diet, and environmental factors. The dynamic population of the gut microbiome, majorly consisting of Bacteroidetes and Firmicutes, forms a complex ecological community. The collective metabolic activities and interaction of gut microbiota with the host exert a marked influence on human physiology. The gut microbiota help to perform various functions, such as maintenance of intestinal mucosal integrity, production of anti-microbial peptides, protection against foreign invaders, and immunity development. In addition, they also provide essential nutrients, such as enzymes and vitamins, and also take part in metabolite synthesis, which influences both cognitive and behavioral functions of the human host. Homeostatic equilibrium among gut microorganisms, and between the microbes and intestinal interface of the host allows the maintenance of beneficial microbiota. Any alteration or dysbiosis in gut microbial composition, due to a sedentary lifestyle or intake of an unbalanced diet, plays a crucial role in the development of diseases like systemic inflammation, insulin resistance, auto-immune and metabolic disorders. This review summarizes our current understanding of the gut microbial organization, its importance in the fundamental biological processes and the pathogenesis of various human diseases and infections, and also the prognostic, diagnostic, and therapeutic potential of the gut microbiota.
Yeast is widely used for industrial production of various types of products, such as ethanol and enzymes. However, its fermentation efficiency is strongly reduced by harmful environmental stresses. Specifically, harmful environmental stresses damage important cellular components, such as cell wall, cell membrane, proteins, etc. Then, these damages cause cellular metabolic disorders or even death. In the past decades, there has been a portfolio of studies on the environmental stress tolerance of yeasts, which mainly aimed at cell damages caused by different environmental stresses, different ways to improve yeast environmental stress tolerance or a tolerance mechanism for certain environmental stress. However, a comprehensive overview of how yeasts respond to environmental stresses is lacking, and the correlation of tolerance mechanism between different environmental stresses is unclear. In this review, we summarized the general damages induced by most of environmental stresses, the existing major mechanisms of environmental stress tolerance from the perspective of key signalling pathways, and the common ways to improve the resistance to environmental stresses in yeast cells. The tolerance mechanisms of yeast cells to different environmental stresses are diverse, but sometimes they share the same signalling pathway. Cells use sensors on the cell surface to recognize environmental stresses and transmit signals to the nucleus to cause changes in gene expression. By summarizing the main signalling pathways, including MAPK pathway, cAMP/PKA pathway, YAP1/SKN7 pathway, it will provide a powerful reference for future efforts to promote yeast environmental stress tolerance and study yeast tolerance mechanisms.
Lignocellulose is an abundant and renewable biomass that is mainly composed of cellulose, hemicellulose and lignin. The development and utilization of lignocellulosic ethanol is an effective way to solve the energy problem/crises, In addition, lignocellulosic ethanol industry can play a significant role in controlling environmental pollution and extending agricultural industrial chain; however, it has not been able to realize large-scale industrialization due to the limitation of both biotechnology and economy. This review first introduces industrialization status of lignocellulosic ethanol, and then focuses on the progress of practical technology and analyzing the economic feasibility of the second-generation bioethanol plant; finally, the future development trend of the industry was prospected. To summarize, continuous technological innovation is needed in vital steps such as pretreatment, enzymatic hydrolysis, and fermentation. Meanwhile, for making the cellulosic ethanol industry truly economically competitive, we also need to achieve interdisciplinary, cross-domain integration innovation, such as the construction of raw material collection and storage system, in situ producing special enzyme system, high-value utilization of raw material components, developing a closely integration of biomass refinery with mature equipment and overall industrialization programs, etc. It is hoped that this review can provide a useful reference for the industrialization of second-generation bioethanol.
The complex mechanisms of the internal operation of cellular functions have not been fully resolved and these functions are regulated by multiple effects, such as transcription regulation, signal transduction, and enzyme catalysis, forming complex interactive mechanisms. This makes the construction of a whole-cell computational model, containing various complex cellular functions, very challenging. However, biological models have played a significant role in the field of systems biology, such as guiding gene-target mining and studying cell metabolic characteristics. Therefore, there is increasing research interest in the construction of whole-cell computational models. Combining two classical languages of systems biology, this review expounds on the development and challenges of whole-cell computational modeling from the two classical methods of steady-state and dynamic modeling. Finally, we propose a new approach for constructing whole-cell computational models.
Biogenic amines (BAs) are potential amine hazards that are detected in fermented foods and alcoholic beverages. Excessive intake of BAs may lead to allergic symptoms such as difficulty in breathing, nausea, and vomiting. Degradation of BAs by multicopper oxidase (MCO) is a promising method as it has little effect on the fermentation process, food nutrition, and flavor. However, the application of MCO in food industry was restricted due to its poor catalytic properties and low productivity. In this work, food-grade expression of the Bacillus amyloliquefaciens MCO (MCOB) and its three mutants were successfully constructed in Lactococcus lactis NZ3900. The expression level of MCOB in L. lactis NZ3900 was dramatically enhanced by optimizing the cultivation conditions, and the highest expression level reached 4488.1 U/L. This was the highest expression level of food-graded MCO reported so far, to our knowledge. Interestingly, the optimal reaction pH of MCOB expressed in L. lactis NZ3900 switched to 4.5, it would be more suitable for degrading BAs in food as the pH value of most fermented foods was found to be 4.5. Moreover, MCOB expressed in L. lactis NZ3900 was quite stable (with more than 80% residual activity) in the pH range of 4.0–5.5, the catalytic rate constant (k cat) and specific activity of MCOB LS were all dramatically increased compared with that of MCOB expressed in Escherichia coli. Using histamine as the substrate, the degradation of BAs within 24 h by MCOB expressed in L. lactis NZ3900 was 69.7% higher than that expressed in E. coli. The results demonstrated the potential applications of MCOB in food industry for reduction of biogenic amines.
Glucose oxidase (GOD) has many practical applications, but its poor thermostability limits its broader use. In this research, three primary mutants of wild-type GOD were designed using rational mutagenesis, and the GODm mutant was constructed by combinatorial design. The expression, purification, and enzymatic properties of the mutants were studied. The specific enzyme activity of GODm was 2.10-fold higher than that of wild type, and the (k cat/K m) value was increased by 1.45-fold. After treatment at 55 ℃ for 3 h, GODm retained 37.5% of its enzymatic activity, and the half-life (t 1/2) of GODm at 55 ℃ and 65 ℃ was 2.28-fold and 3.36-fold higher than that of wild type, respectively. By analyzing the three-dimensional structure of wild type and the GODm mutant, it was found that T30V formed a new hydrogen bond with FAD and strengthened the hydrophobic interaction, D315K optimized the surface electrostatic interaction, and A162T improved the efficiency of the electron pathway. Thus, a novel mutant with improved thermostability and catalytic efficiency was obtained in this research.
Gamma-aminobutyric acid is an important nonprotein amino acid and has been extensively applied in pharmaceuticals, livestock, food additives, and so on. It is important to develop Corynebacterium glutamicum strains that can efficiently produce gamma-aminobutyric acid from glucose. In this study, production of gamma-aminobutyric acid in C. glutamicum CGY700 was improved by construction of CO2 anaplerotic reaction and overexpression of citrate synthase. The co-expression of ppc encoding phosphoenolpyruvate carboxylase and gltA encoding citrate synthase was constructed and optimized in the chromosome to compensate carbon loss and conquer metabolic bottleneck. The expression of ppc and gltA were controlled by promoters Ptac and PtacM, and the optimal mode of PtacM-ppc-Ptac-gltA was determined. Simultaneously, the genes pknG encoding serine/threonine protein kinase G and ldh encoding l-lactate dehydrogenase were deleted, and glnA2 encoding glutamine synthase was overexpressed in the chromosome. The final strain CGY-PG-304 constructed in this study could produce 41.17 g/L gamma-aminobutyric acid in shake flask cultivation and 58.33 g/L gamma-aminobutyric acid via Fed-Batch fermentation with a yield of 0.30 g/g glucose. CGY-PG-304 was constructed by genome editing; therefore, it is stable and not necessary to add any antibiotics and inducer during fermentation.
Multispecies solid-state fermentation is a traditional processing technique for the traditional Chinese food, such as cereal vinegar, Baijiu, etc. Generally, few abundant and many rare microbes were involved in such processes, and the necessity and roles of the latter are less studied. Here the co-occurrence patterns of abundant and rare bacterial community and abiotic factors influencing their community assembly were investigated in acetic acid fermentation following starter inoculation, using Zhenjiang aromatic vinegar as a model system. Abundant taxa that contribute to the function of accumulating acid exhibited a ubiquitous distribution while the distribution of rare taxa along the fermentation process unraveled. The species composition of the rare taxa significantly altered, but abundant taxa were maintained after inoculation. Moreover, the diversity of rare taxa changed more significantly than that of abundant taxa. Both abundant and rare sub-communities, which were contributed more with species turnover than species richness, were demonstrated to be driven by pH, acetic acid, ammonium nitrogen, and ethanol. Stochastic processes regulated the assembly of both sub-communities, but more prominent on rare sub-communities. Co-occurrence network was more governed by rare sub-communities, and the co-variations between microbial communities were predominantly positive, implying that rare taxa played more important role in the fermentation stability and network robustness. Furthermore, seven network connectors were identified, and three of them belonged to rare taxa. These microbes of different modules were enriched at particular phases of fermentation. These results demonstrate the ecological significance of rare bacteria and provide new insights into understanding the abiotic factors influence microbial structure in traditional fermented foods.
Succinate is an important building block for chemical synthesis. However, during the fermentation process, excessive osmotic stress and byproduct accumulation substantively impair the performance of the microbial cell factory. To this end, two strategies were proposed. First, an osmo-tolerant mutant, Escherichia coli FMME-N-2, was screened by combined mutagenesis (ARTP and 60Co-γ irradiation) to produce 51.8 g L−1 succinate with a productivity of 0.81 g L−1 h−1. Second, an oxygen-dependent bifunctional switch (OBS) was developed with promoter PfnrF8-based activation and tobacco etch virus protease-based inhibition functions. With ribosomal binding site (RBS) and degron optimization of OBS, the optimal strain E. coli FMME-N-30 achieved a succinate titer and productivity of 119 g L−1 and 1.65 g L−1 h−1, respectively, in a 30-L fermentor, while only 7.1 g L−1 acetate and no formate or lactate were detected. Compared to the wild-type strain E. coli FMME-N, the succinate titer was increased by 3.3-fold. These results highlight the applicability of OBS for the large-scale production of value-added chemicals.
The natural concentration of bovine lactoferrin C-lobe is low and its separation by proteolytic enzyme digestion is difficult. Here, we expressed the codon-optimized fragment of C-lobe on plasmid pMA0911 with the P veg promoter in Bacillus subtilis 168 at 20 °C. The yield was 7.5 mg/L, and 90.6% purity was achieved using ammonium sulfate precipitation, Ni–NTA and molecular exclusion. The C-lobe at 10 mg/mL completely inhibited cell growth of Escherichia coli JM109 (DE3) and Pseudomonas aeruginosa CGMCC 1.6740, and 48.4% of growth of Staphylococcus aureus CGMCC 1.282, the result is similar to that of 200 ng/mL N-lobe. The minimum inhibitory concentrations of C-lobe were 4, 8 and 16 mg/mL, while those of N-lobe were 128, 256 and 512 μg/mL for E. coli, P. aeruginosa and S. aureus, respectively. This is the first report on bovine lactoferrin C-lobe expression and the comparative resistance of the recombinant N- and C-lobes in a food-safe strain of B. subtilis. Our findings offer the potential to study the structure–function relationship of the N- and C-lobes recombinantly produced in the same host.