The anaerobic fermentation of crude glycerol to 1,3-propanediol by Clostridium butyricum is energetically favorable due to the low stirring rate and unsterilized fermentation environment. However, its efficiency is severely limited by the accumulation of acid by-products. Although metabolic engineering is commonly employed to overcome such challenges, the inherent oxygen sensitivity of C. butyricum significantly hinders genetic modification and metabolic analysis under the required conditions. Therefore, we adopted an alternative strategy of supplementing renewable materials to regulate by-products formation and improve the 1,3-propane-diol concentration. Among them, chitosan could reduce the concentration of most by-products, increasing the concentration and yield of 1,3-propanediol to 53.55 g/L and 0.57 g/g. At the same time, activated carbon could achieve co-production of 1,3-propanediol (62.12 g/L) and lactic acid (38.74 g/L) through selective adsorption of by-products. Combined with the adsorption equation-assisted dynamic metabolic flux analysis developed in this work, we demonstrated the by-product regulation produces more NADH and brings dynamic balance to the reducing power. This work offers enlightenment and reference for a similar production process, which is of great potential to reduce the concentration of most by-products in future large-scale 1,3-propanediol production.

Prebiotics are selectively utilized substrates that modulate gut microbiota and host health, yet different prebiotic structures may elicit distinct ecological and metabolic responses. In this study, we investigated the effects of five structurally diverse prebiotics—isomaltooligosaccharides (IMO), arabinogalactans (AG), pectin, inulin, and stachyose—on human gut microbiota via a 24 h in vitro anaerobic culture with healthy donors’ gut microbiota. Microbial community dynamics were profiled by 16S rRNA gene sequencing, and short-chain fatty acids (SCFAs) production was analyzed. All treatments resulted in decreased α-diversity compared with baseline, with pectin most effectively preserving microbial richness and evenness, whereas stachyose led to the greatest reduction. Community composition and functional profiles shifted in a substrate-specific manner, with AG promoting Bacteroidaceae, IMO stimulating Lachnospiraceae and Faecalibacterium, and pectin supporting balanced microbial structures and SCFA production. Pectin, IMO, and inulin enhanced butyrate levels, whereas AG and pectin promoted propionate formation. These findings demonstrate that prebiotic structural differences strongly shape gut microbial ecology and metabolism, providing a mechanistic basis for rationally selecting and combining prebiotics to beneficially modulate the gut microbiota.

Zearalenone (ZEN) is one of the most prevalent mycotoxins in the world, with estrogenic toxicity leading to significant annual economic losses and environmental pollution. RmZHD, a novel ZEN hydrolase, surpasses the efficiency of its predecessors but faces challenges in large-scale industrial application. In this work, an engineered Escherichia coli that can degrade zearalenone is constructed based on synthetic biology and surface display methods. It can degrade 94% of zearalenone at 30 °C in 1 h (the final concentration was 1.898 μg/mL) and effectively degrade ZEN-derived toxins, including zearalanone, β-zearalanol, α-zearalenol, and β-zearalenol. This engineered E. coli requires no additional manipulation for the surface display of RmZHD and is cost-effective to produce. Moreover, it exhibits the capability to degrade ZEN in maize feed while concurrently mitigating inflammation in animal reproductive and digestive organs. In summary, the engineered E. coli with surface-displayed RmZHD presents a novel approach for environmentally sustainable and industrial-scale treatment of ZEN.

With the advancement of science, technology, and productivity, the rapid development of industrial production, transportation, and the exploitation of fossil fuels has gradually led to the accumulation of greenhouse gases and deterioration of global warming. Carbon neutrality is a balance between absorption and emissions achieved by minimizing carbon dioxide (CO2) emissions from human social productive activity through a series of initiatives, including energy substitution and energy efficiency improvement. Then CO2 was offset through forest carbon sequestration and captured at last. Therefore, efficiently reducing CO2 emissions and enhancing CO2 capture are a matter of great urgency. Because many species have the natural CO2 capture properties, more and more scientists focus their attention on developing the biological carbon sequestration technique and further combine with synthetic biotechnology and electricity. In this article, the advances of the synthetic biotechnology method for the most promising organisms were reviewed, such as cyanobacteria, Escherichia coli, and yeast, in which the metabolic pathways were reconstructed to enhance the efficiency of CO2 capture and product synthesis. Furthermore, the electrically driven microbial and enzyme engineering processes are also summarized, in which the critical role and principle of electricity in the process of CO2 capture are canvassed. This review provides detailed summary and analysis of CO2 capture through synthetic biotechnology, which also pave the way for implementing electrically driven combined strategies.

Naringinase is a kind of glycosidase that catalyzes the biotransformation of naringin into naringenin. It is a multi-compound enzyme that provides the activity of alpha-L-rhamnosidase and beta-glucosidase. In this study, an artificial naringinase system was constructed by co-immobilizing alpha-L-rhamnosidase (Aspergillus oryzae FJ0123, Ao-rha) and beta-glucosidase (Thermotoga maritima MSB8, Tm-glu) on magnetic silica-based chitosan microspheres (MSC). The molar ratio of enzymes and the preparation conditions were optimized for improving synergistic effect and immobilization efficiency. Under optimized conditions (the molar ratio of Ao-rha and Tm-glu, 3:1; temperature, 25 degrees C; glutaraldehyde concentration, 2.0%; pH, 3.0; time, 9 h), the immobilization yield and activity recovery were 61.4% and 37.3% for Ao-rha and 90.1% and 56.3% for Tm-glu, respectively. Biochemical characterization indicated that MSC-naringinase had better adaptability and stability than free enzymes. The activity of MSC-naringinase was maintained at 58.7% after 10 times of recycling. The catalytic conditions were also investigated. The cascade reactions of naringin to prunin and prunin to naringenin by using MSCnaringinase resulted in a yield of 0.62 mg/mL and a conversion rate of 96.9%, respectively, without prunin accumulation. These results indicated the great potential of MSC-naringinase as an efficient artificial naringinase system for cooperative enzymatic synthesis of naringenin.

[This corrects the article DOI: 10.3389/fnut.2022.851402.].

The intestine is a potential location for berberine (BBR) to exert its therapeutic effects, but the understanding of the influences of BBR on the gut microbiota is limited. Through in vitro fermentation of human intestinal microbiota, we investigated the effects of BBR on microbiota composition and metabolism. The result indicated that BBR reduced the production of acetic acid and propionic acid and had no effect on the content of butyric acid. Analysis of the 16S rRNA gene-based community revealed that BBR increased the abundance of Faecalibacterium and decreased the abundance of Bifidobacterium, Streptococcus and Enterococcus. Through metabolomics analysis, BBR treatment regulated various amino acid metabolism pathways of intestinal microbiota, especially tyrosine, serine and L-glutamate. Our study presented direct impacts of BBR on the intestinal microbiota, which provided the probable targets of the therapies by BBR and supported further exploration of the underlying mechanisms.

Oxidoreductases play an important role in the metabolism of life so that revealing their catalytic mechanism is momentous for further reveal the influence of electron and proton transfer on the growth of living organisms. More importantly, oxidoreductase accounts for about one-third among all enzymes newly registered on the BRENDA website, of which half using NAD(P)H/NAD(P)(+) as a coenzyme. Hugo Theorell is awarded a Nobel Prize in 1955 for achievements that discovered the nature and mode of action of oxidation enzymes in 1951. Cooperate with Britton Chance, they propose the famous Theorell-Chance mechanism based on the research of the catalysis kinetics mechanism of liver alcohol dehydrogenase, which has always been a hot topic since then. Thus, in the past 70 years, the study of the mechanism and its modification has always been a focus of researches since the classic oxidoreductase's catalysis mechanism is put forward in 1951. Based on the previous work, we review the evolution of the catalytic mechanism of oxidoreductases systematically and thus put forward two valuable scientific problems. (1) Do intracellular coenzymes bound to oxidoreductases enzymes have to dissociate after oxidation or reduction? (2) Do intracellular NAD(P)H-dependent oxidoreductases enzyme and its corresponding coenzyme have self-assembly? Then, we also detailed introduce some discoveries of oxidoreductases catalytic mechanism based on our recent work. Based on the whole-cell catalytic process of 1,3-propanediol oxidoreductase and glycerol dehydrogenase, we find that the whole-cell can also catalyze the extracellularly NAD(+) to NADH. After then, technology development brings us advanced precise instruments, which give us a deeper insight into the catalyzation process at a single-molecular level. Among them, the STM-BJ (scanning tunneling microscope break junction) technique provides excellent performance in spatial resolution and electrical detection sensitivity, which has been one of the most frequently used techniques in single-molecule electrical detection in recent years. Thus, we provide the first demonstration of the STM-BJ technique for investigating charge transport through a single active enzyme junction, in which the binding of NAD(+) with proteins boosts the charge transport by over 2100% than neutral FDH. Combine with site-specific mutagenesis, we demonstrate the conductance of FDH-NAD(+) highly correlate with their bioactivities. And we will also employ it to study the real-time conductivity of single NAD(P)H-depending formate dehydrogenase in the catalytic process to reveal the new catalytic mechanism at the single-molecule level in future work. Studying the combination of single NAD(P)H-depending oxidoreductase and its co-enzyme and the reaction of substrate catalyzing is an original innovative research, which will help us to understand the essence of enzyme catalyzation in life activity and to improve technologies in directed enzyme evolution and development of biomedical drugs. The proposed catalytic mechanism of the NAD(P)H-depending oxidoreductase is expected not only to revolutionize the theory that has been followed for 70 years but also to improve a wide range of traditional technologies, such as whole-cell fermentation, multiple enzymes coupling catalyzation, enzyme activity detection, bio-sensor and so on.

NAD(P)H-dependent enzymes are ideal biocatalysts for the industrial production of chiral compounds, such as chiral alcohols, chiral amino acids, and chiral amines; however, efficient strategies for the regeneration of coenzyme are expected as costly of the coenzymes. Herein, a solvent-tolerant isopropanol dehydrogenase (IDH) showing lower similarity (37%) with other proteins was obtained and characterized. The enzyme exhibits high catalysis ability of its substrates methanol, ethanol, ethylene glycol, glycerol, isopropanol, n-butanol, isobutanol, and acetone. And it has good adaptability in organic solvents (isopropanol, acetonitrile, acetone, and acetophenone). Interaction force and the corresponding amino acid residues between IDH and NAD(+) or NADP(+) were parsed by docking. The wide substrate spectrum, excellent organic solvent tolerance, and good biocatalytic activity make the excavated enzyme a promising biocatalyst for the production of chiral compounds industrially and the construction of coenzyme regeneration systems in aqueous organic phase or organic phase.

With the aggravation of environmental pollution and energy crisis, the sustainable microbial fermentation process of converting glycerol to 1,3-propanediol (1,3-PDO) has become an attractive alternative. However, the difficulty in the online measurement of glycerol and 1,3-PDO creates a barrier to the fermentation process and then leads to the residual glycerol and therefore, its wastage. Thus, in the present study, the four-input artificial neural network (ANN) model was developed successfully to predict the concentration of glycerol, 1,3-PDO, and biomass with high accuracy. Moreover, an ANN model combined with a kinetic model was also successfully developed to simulate the fed-batch fermentation process accurately. Hence, a soft sensor from the ANN model based on NaOH-related parameters has been successfully developed which cannot only be applied in software to solve the difficulty of glycerol and 1,3-PDO online measurement during the industrialization process, but also offer insight and reference for similar fermentation processes.

Clostridium butyricum is one of the best 1,3-propanediol producers due to the nonpathogenic, less byproducts, and energy-efficient fermentation process. In fermentation process, the relationship among substrate, product, and byproducts is intricate and hard to be analyzed. The present study is aimed at establishing a novel kinetic model not only based on biomass, substrate, and 1,3-propanediol, but also considering the byproduct concentration to describe 1,3-propanediol fermentation process by C. butyricum. The simulative result of the model fit well with that in the batch fermentation process. Furthermore, the model was also used to predict the result of fed-batch fermentation process after some modifications. The predicted result of model fit well with the data in experiment when glycerol was controlled at around 10 g/L. Thus, this novel kinetic model could serve as a tool for further optimization of the fermentation process, and could be improved for some other similar processes.

A bioinspired strategy for the synthesis of supramolecular and biocatalytical materials was developed base on protein-protein supramolecular interaction and genetic engineering. Formate dehydrogenase (FDH) and its functional fragments were separately fused to form a multi-function domain. The fusion proteins and functional fragments self-assembled into the expanded and controllable supramolecular interaction networks. Morphology characterization by scanning-electron microscopy showed that the assembled functional fragments and fusion proteins formed multi-dimensional (3D) and two-dimensional (2D) layer-like structures. Moreover, the oligomeric biocatalysts exhibited higher structural stability and NAD(H) recycling efficiency than the unassembled structures when they were applied to a co-enzyme regeneration system. These results suggest that the bioinspired strategy provides a promising approach for the fabrication of supramolecular FDH materials via genetic engineering and self-assembly. The significant improvement on the biocatalytical activity reveals the essential role of supramolecular interface design in their biocatalysis applications.

In order to obtain the excellent 1,3-propanediol (1,3-PDO) producer from wild-type Clostridium butyricum, adaptive evolution was carried out to select the strain for fast growth. The most significant change was that fermentation time decreased from 36 h to 20 h after adaptive evolution. Thus, it led to the corresponding volumetric productivity of 1,3-PDO increasing from 0.97 to 2.14 (g/L center dot h(-1)) which increased by 114%. Adaptive evolution was also applied to butyric acid tolerant strain selected based on the fast-growing one through a simple equipment. 1,3-PDO concentration increased from 40.28 to 66.23 g/L through fed-batch fermentation by butyric acid tolerant strain compared with the fast-growing one. In addition, the endpoint strain was successfully and steadily used in the 50-L scale fermentation. Thus, adaptive evolution is an excellent strategy which can help us select the fast-growing strain and reduce the negative effect from substrate and metabolite inhibition. (c) 2018 American Institute of Chemical Engineers AIChE J, 65: 32-39, 2019

The human intestinal microbiota has an important role in the maintenance of human health and disease pathogenesis. The aim of this research was to investigate the impact of four media on human intestinal microbiota metabolite and composition changes, we performed in vitro batch culture using intestinal microbiota samples from three fecal microbiota transplantation (FMT) donors. After 48 h culture, gut microbiota medium (GMM) had the highest production of acetic acid (73.00 ± 7.56 mM) and propionic acid (16.79 ± 1.59 mM), bacterial growth media (BGM) had the highest production of butyric acid (13.39 ± 0.56 mM). In addition, brain heart infusion (BHI) promoted (p < 0.05) the growth of Bacteroidetes, especially Bacteroides after 48 h, GMM resulted in a significant increase (p < 0.05) in Actinobacteria and increased the beneficial genus Bifidobacterium, fastidious anaerobe broth (FAB) increased Firmicutes population, and BGM promoted the growth of Escherichia–Shigella and Akkermansia. The results suggest that four media had different effects on the human intestinal microbiota metabolism and composition in vitro. These results may facilitate the culture of bacteria from the human intestinal microbiota.

Collagen is widely distributed in various tissues of living organisms and is one of the basic proteins that constitute living organisms. The structure of collagen is more complicated than that of general protein and it displays a more stable property so that normal proteases have no significant effect to deal with it. There are only two typical enzymes have ability to hydrolyze collagen in situ , one is called matrix metalloproteinase (MMP) and another is bacterial collagenase. In this review the collagen and hydrolase are introduced in briefly, then the development process and the hydrolysis mechanism to collagen between matrix metalloproteinase and collagenase are presented, then the particular aspects of the different enzyme activities will be contextualized within relevant areas of application, mainly about therapeutics, food processing, environmental protection and tissue engineering. In the end the development of the present research and guide further potential research orientation of the system of collagen&collagenase is summarized.

As phenylalanine dehydrogenase (PheDH) plays an important role in the synthesis of chiral drug intermediates and detection of phenylketonuria, it is significant to obtain a PheDH with specific and high activity. Here, a PheDH gene, pdh, encoding a novel BhPheDH with 61.0% similarity to the known PheDH from Microbacterium sp., was obtained. The BhPheDH showed optimal activity at 60 °C and pH 7.0, and it showed better stability in hot environment (40–70 °C) than the PheDH from Nocardia sp. And its activity and thermostability could be significantly increased by sodium salt. After incubation for 2 h in 3 M NaCl at 60 °C, the residual activity of the BhPheDH was found to be 1.8-fold higher than that of the control group (without NaCl). The BhPheDH could tolerate high concentration of ammonium chloride and its activity could be also enhanced by the high concentration of ammonium salts. These characteristics indicate that the BhPheDH possesses better thermostability, ammonium chloride tolerance, halophilic mechanism, and high salt activation. The mechanism of thermostability and high salt tolerance of the BhPheDH was analyzed by molecular dynamics simulation. These results provide useful information about the enzyme with high-temperature activity, thermostability, halophilic mechanism, tolerance to high concentration of ammonium chloride, higher salt activation and enantio-selectivity, and the application of molecular dynamics simulation in analyzing the mechanism of these distinctive characteristics.

1,3-propanediol production by Clostridium butyricum is a low productivity process due to the long time seed cultivation and thus hinders its industrial scale production. In the present study, repeated batch fermentation coupled with activated carbon adsorption strategy was first established which conduced not only to saving the time of seed cultivation and enhancing the productivity, but also to reducing the costs for the seed cultivation to achieve the purpose of 1,3-propanediol continuous production. The concentration of 1,3-propanediol from first to fourth cycle was 42.89, 45.78, 44.48, 42.39 (g/L), and the corresponding volumetric productivity was 2.14, 1.91, 1.85, 2.12 (g/L · h-1 ) respectively. More importantly, a relatively complete schematic diagram of the proposed metabolic pathways was firstly mapped out based on the intracellular metabolites analysis through GC-MS. At the same time, metabolic pathway and principal components analyses were carried out to give us deep insight into metabolic state. Many metabolites occurred to response to the stress in Cycle II. Even resting body formed and lipid accumulated owing to the worsening environment in the group without activated carbon in Cycle III. Thus, it demonstrated that activated carbon provided a favorable microenvironment for Clostridium butyricum in the repeated batch fermentation process to achieve the purpose of 1,3-propanediol continuous production.

This study reports the application of peptide linker in the construction of bifunctional formate dehydrogenase (FDH) and leucine dehydrogenase (LeuDH) enzymatic complex for efficient cofactor regeneration and L-tert leucine (L-tle) biotransformation. Seven FDH-LeuDH fusion enzymes with different peptide linker were successfully developed and displayed both parental enzymeactivities. The incorporation order of FDH and LeuDH was investigated by predicting three-dimensional structures of LeuDH-FDH and FDH-LeuDH models using the I-TASSER server. The enzymatic characterization showed that insertion of rigid peptide linker obtained better activity and thermal stability in comparison with flexible peptide linker. The production rate of fusion enzymatic complex with suitable flexible peptide linker was increased by 1.2 times compared with free enzyme mixture. Moreover, structural analysis of FDH and LeuDH suggested the secondary structure of the N-, C-terminal domain and their relative positions to functional domains was also greatly relevant to the catalytic properties of the fusion enzymatic complex. The results show that rigid peptide linker could ensure the independent folding of moieties and stabilized enzyme structure, while the flexible peptide linker was likely to bring enzyme moieties in close proximity for superior cofactor channeling.

Multi-enzyme complexes have the potential to achieve high catalytic efficiency for sequence reactions due to their advantages in eliminating product inhibition, facilitating intermediate transfer and in situ regenerating cofactors. Constructing functional multi-enzyme systems to mimic natural multi-enzyme complexes is of great interest for multi-enzymatic biosynthesis and cell-free synthetic biotransformation, but with many challenges. Currently, various assembly strategies have been developed based on the interaction of biomacromolecules such as DNA, peptide and scaffolding protein. On the other hand, chemical-induced assembly is based on the affinity of enzymes with small molecules including inhibitors, cofactors and metal ions has the advantage of simplicity, site-to-site oriented structure control and economy. This review summarizes advances and progresses employing these strategies. Furthermore, challenges and perspectives in designing multi-enzyme systems are highlighted.