Rewiring Bacillus subtilis and bioprocess optimization for oxidoreductive reaction-mediated biosynthesis of D-tagatose.

D-tagatose holds significant importance as a functional monosaccharide with diverse applications in food, medicine, and other fields. This study aimed to explore the oxidoreductive pathway for D-tagatose production, surpassing the contemporary isomerization-mediated biosynthesis approach in order to enhance the thermodynamic equilibrium of the reactions. Initially, a novel galactitol dehydrogenase was discovered through biochemical and bioinformatics analyses. By co-expressing the galactitol dehydrogenase and xylose reductase, the oxidoreductive pathway for D-tagatose synthesis was successfully established in Bacillus subtilis. Subsequently, pathway fine-tuning was achieved via promoter regulation and dehydrogenase-mediated cofactor regeneration, resulting in 6.75-fold higher D-tagatose compared to that produced by the strain containing the unmodified promoter. Finally, optimization of fermentation conditions and medium composition produced 39.57 g/L D-tagatose in a fed-batch experiment, with a productivity of 0.33 g/L/h and a yield of 0.55 mol/mol D-galactose. These findings highlight the potential of the constructed redox pathway as an effective approach for D-tagatose production.

Efficient biosynthesis of 3-hydroxypropionic acid from glucose through multidimensional engineering of Escherichia coli.

3-Hydroxypropionic acid (3-HP) is a top value-added chemical with multifaceted application in chemical, material, and food field. However, limited availability of robust strains and elevated fermentation costs currently impose constraints on sustainable biosynthesis of 3-HP. Herein, transporter engineering, metabolic dynamic modulation, and enzyme engineering were combined to address above limitations. First, a glucose-utilizing 3-HP biosynthetic pathway was constructed in Escherichia coli, followed by recruiting alternative glucose transport system to overcome center metabolism overflow. Next, the Cra (a transcription factor)-dependent switch was applied to autonomously fine-tune carbon flux, which alleviated growth retardation and improved the 3-HP production. Subsequently, inactivation of glycerol facilitator (GlpF) increased intracellular glycerol levels and boosted 3-HP biosynthesis, but caused toxic intermediate 3-hydroxypropionaldehyde (3-HPA) accumulation. Furthermore, semi-rational design of aldehyde dehydrogenase (YdcW) increased its activity and eliminated 3-HPA accumulation. Finally, fed-batch fermentation of the final strain resulted in 52.73 g/L 3-HP, with a yield of 0.59 mol/mol glucose.

Functional and Structural Roles of the Dimer Interface in the Activity and Stability of Clostridium butyricum 1,3-Propanediol Oxidoreductase.

Biosynthesis of 1,3-propanediol (1,3-PD) by 1,3-propanediol oxidoreductase (PDOR) is often limited by the stability issues. To address this issue, the goal of the present study was to engineer the Clostridium butyricum PDOR dimeric interface. The interface exists between the chains and plays a role in the synthesis of 1,3-PD, which is hindered by the increased temperature and pH. Herein, we engineered PDOR by HotSpot Wizard 3.0 and molecular dynamics simulations, improving its thermal stability, pH tolerance, and catalytic properties with respect to the wild-type PDOR activity at 37 °C. Compared to the activity of the wild-type PDOR, the N298C mutant showed 0.5-fold greater activity at pH 8.0, while the P299E mutant showed significantly increased activity of over five fold at pH 4.0. Further structural comparisons between the wild-type and P299E mutant revealed that the extraordinary stability of the P299E mutant could be due to the formation of additional hydrogen bonds and salt bridges. The N298C mutant also exhibits thermal stability at a broad range of temperature at pH 8 with respect to wild-type PDOR and other mutants. The molecular dynamics simulations revealed that stability profiles of P299E mutants at pH 4.0 are attributed to identical root mean square deviation values and stable conformations in the motif region present in the dimer interface of the enzyme. These findings suggest that the dimer interface motifs are essential for the compactness and stability of the PDOR enzyme; therefore, engineering the PDOR using a structure-guided approach could aid in improving its activity and stability under various physiological conditions (pH and temperature).

Co-fermentation of glycerol and glucose by a co-culture system of engineered Escherichia coli strains for 1,3-propanediol production without vitamin B12 supplementation.

The necessity of costly co-enzyme B12 for the activity of glycerol dehydratase (GDHt) is considered as a major bottleneck in sustainable bioproduction of 1,3-propanediol (1,3-PD) from glycerol. Here, an E. coil Rosetta-dhaB1-dhaB2 strain was constructed by overexpressing a B12-independent GDHt (dhaB1) and its activating factor (dhaB2) from Clostridium butyricum. Subsequently, it was used in designing a co-culture with E. coli BL21-dhaT that overexpressed 1,3-PD oxidoreductase (dhaT), to produce 1,3-PD during co-fermentation of glycerol and glucose. The optimum initial ratio of BL21-dhaT to Rosetta-dhaB1-dhaB2 strains in the co-culture was 1.5. Compared to the fermentation of glycerol alone, co-fermentation approach provided 1.3-folds higher 1,3-PD. Finally, co-fermentation was done in a 10 L bioreactor that produced 41.65 g/L 1,3-PD, which corresponded to 0.69 g/L/h productivity and 0.67 mol/mol yield of 1,3-PD. Hence, the developed co-culture could produce 1,3-PD cost-effectively without requiring vitamin B12.

Biocatalytic Synthesis of D-Allulose Using Novel D-Tagatose 3-Epimerase From Christensenella minuta.

D-allulose, which is one of the important rare sugars, has gained significant attention in the food and pharmaceutical industries as a potential alternative to sucrose and fructose. Enzymes belonging to the D-tagatose 3-epimerase (DTEase) family can reversibly catalyze the epimerization of D-fructose at the C3 position and convert it into D-allulose by a good number of naturally occurring microorganisms. However, microbial synthesis of D-allulose is still at its immature stage in the industrial arena, mostly due to the preference of slightly acidic conditions for Izumoring reactions. Discovery of novel DTEase that works at acidic conditions is highly preferred for industrial applications. In this study, a novel DTEase, DTE-CM, capable of catalyzing D-fructose into D-allulose was applications. In this study, a novel DTEase, DTE-CM, capable of catalyzing D-fructose into D-allulose was DTE-CM on D-fructose was found to be remarkably influenced and modulated by the type of metal ions (co-factors). The DTE-CM on D-fructose was found to be remarkably influenced and modulated by the type of metal ions (co-factors). The 50°C from 0.5 to 3.5 h at a concentration of 0.1 mM. The enzyme exhibited its maximum catalytic activity on D-fructose at pH 6.0 and 50°C from 0.5 to 3.5 h at a concentration of 0.1 mM. The enzyme exhibited its maximum catalytic activity on -fructose at pH 6.0 and 50°C with a K cat /K m value of 45 mM-1min-1. The 500 g/L D-fructose, which corresponded to 30% conversion rate. With these interesting catalytic properties, this enzyme could be a promising candidate for industrial biocatalytic applications.

Exploring a Highly D-Galactose Specific L-Arabinose Isomerase From Bifidobacterium adolescentis for D-Tagatose Production.

D-Galactose-specific L-arabinose isomerase (L-AI) would have much potential for the enzymatic conversion of D-Galactose into D-tagatose, while most of the reported L-AIs are L-arabinose specific. This study explored a highly D-Galactose-specific L-AI from Bifidobacterium adolescentis (BAAI) for the production of D-tagatose. In the comparative protein-substrate docking for D-Galactose and L-arabinose, BAAI showed higher numbers of hydrogen bonds in D-Galactose-BAAI bonding site than those found in L-arabinose-BAAI bonding site. The activity of BAAI was 24.47 U/mg, and it showed good stability at temperatures up to 65°C and a pH range 6.0-7.5. The K m, V max, and K cat/K m of BAAI were found to be 22.4 mM, 489 U/mg and 9.3 mM-1 min-1, respectively for D-Galactose, while the respective values for L-arabinose were 40.2 mM, 275.1 U/mg, and 8.6 mM-1 min-1. Enzymatic conversion of D-Galactose into D-tagatose by BAAI showed 56.7% conversion efficiency at 55°C and pH 6.5 after 10 h.

Pleiotropic Functions and Biological Potentials of Silver Nanoparticles Synthesized by an Endophytic Fungus.

In recent years, the biological synthesis of silver nanoparticles (AgNPs) from microorganisms has become an emerging trend for developing biocompatible nanomaterials that finds applications in nano and biomedical sectors. In the present study, we demonstrated a facile, green and eco-friendly method for AgNPs synthesis using the endophytic fungi (Colletotrichum incarnatum DM16.3) isolated from medicinal plant Datura metel and its in vitro antithrombin and cytotoxic activity. At first, biosynthesis of colloidal AgNPs was predicted by visual observation of color change and UV-visible spectra demonstrated specific surface plasmon resonance peak at 420 nm which confirmed the presence of nanoparticles. Microscopic analyses revealed the structure of highly aggregated, spherical and crystalline AgNPs in the diameter range of 5-25 nm. Transform infrared spectroscopy (FT-IR) spectral analysis confirmed the presence of probable biomolecules required for the reduction of silver ions. In vitro evaluation of thrombin activity demonstrates that AgNPs could exert strong inhibition against both thrombin activity (87%) and thrombin generation (84%), respectively. Further, in silico based mechanistic analysis yielded a better insight in understanding the probable amino acids responsible for AgNPs binding with thrombin protein. Similarly, in vitro cytotoxicity of synthesized AgNPs on human epithelial cells using MTT assay did not produce any substantial effects after 24 h exposure which indicates excellent biocompatibility nature, whereas notable toxicity was observed on human cancerous (HeLa) cells at 50 µg/mL (IC50 value). In addition, assessment of AgNPs at 10 µg/mL concentration via crystal violet method on biofilm forming Gram-positive (Vibrio cholerae) and Gram-negative bacteria (Bacillus cereus) revealed inhibition up to 85 and 46%, respectively. Overall, this study showed the possibility of microbially synthesized AgNPs as a potent inhibitor for managing acute thrombosis and highlighted their role for other biomedical applications.

New Insights on Cyclization Specificity of Fungal Type III Polyketide Synthase, PKSIIINc in Neurospora crassa.

Type III polyketide synthases (PKSs) biosynthesize varied classes of metabolites with diverse bio-functionalities. Inherent promiscuous substrate specificity, multiple elongations of reaction intermediates and several modes of ring-closure, confer the proteins with the ability to generate unique scaffolds from limited substrate pools. Structural studies have identified crucial amino acid residues that dictate type III PKS functioning, though cyclization specific residues need further investigation. PKSIIINc, a functionally and structurally characterized type III PKS from the fungus, Neurospora crassa, is known to biosynthesize alkyl-resorcinol, alkyl-triketide- and alkyl-tetraketide-α-pyrone products. In this study, we attempted to identify residue positions governing cyclization specificity in PKSIIINc through comparative structural analysis. Structural comparisons with other type III PKSs revealed a motif with conserved hydroxyl/thiol groups that could dictate PKSIIINc catalysis. Site-directed mutagenesis of Cys120 and Ser186 to Ser and Cys, respectively, altered product profiles of mutant proteins. While both C120S and S186C proteins retained wild-type PKSIIINc product activity, S186C favoured lactonization and yielded higher amounts of the α-pyrone products. Notably, C120S gained new cyclization capability and biosynthesized acyl-phloroglucinol in addition to wild-type PKSIIINc products. Generation of alkyl-resorcinol and acyl-phloroglucinol by a single protein is a unique observation in fungal type III PKS family. Mutation of Cys120 to bulky Phe side-chain abrogated formation of tetraketide products and adversely affected overall protein stability as revealed by molecular dynamics simulation studies. Our investigations identify residue positions governing cyclization programming in PKSIIINc protein and provide insights on how subtle variations in protein cores dictate product profiles in type III PKS family.

Novel Type III Polyketide Synthases Biosynthesize Methylated Polyketides in Mycobacterium marinum.

Mycobacterial pathogenesis is hallmarked by lipidic polyketides that decorate the cell envelope and mediate infection. However, factors mediating persistence remain largely unknown. Dynamic cell wall remodeling could facilitate the different pathogenic phases. Recent studies have implicated type III polyketide synthases (PKSs) in cell wall alterations in several bacteria. Comparative genome analysis revealed several type III pks gene clusters in mycobacteria. In this study, we report the functional characterization of two novel type III PKSs, MMAR_2470 and MMAR_2474, in Mycobacterium marinum. These type III pkss belong to a unique pks genomic cluster conserved exclusively in pathogenic mycobacteria. Cell-free reconstitution assays and high-resolution mass spectrometric analyses revealed methylated polyketide products in independent reactions of both proteins. MMAR_2474 protein exceptionally biosynthesized methylated alkyl-resorcinol and methylated acyl-phloroglucinol products from the same catalytic core. Structure-based homology modeling, product docking, and mutational studies identified residues that could facilitate the distinctive catalysis of these proteins. Functional investigations in heterologous mycobacterial strain implicated MMAR_2474 protein to be vital for mycobacterial survival in stationary biofilms. Our investigations provide new insights into type III PKSs conserved in pathogenic mycobacterial species.