Constructing Protein-Scaffolded Multienzyme Assembly Enhances the Coupling Efficiency of the P450 System for Efficient Daidzein Biosynthesis from (2S)-Naringenin.

Daidzein is a major isoflavone compound with an immense pharmaceutical value. This study applied a novel P450 CYP82D26 which can biosynthesize daidzein from (2S)-naringenin. However, the recombinant P450 systems often suffer from low coupling efficiency, leading to an electron transfer efficiency decrease and harmful reactive oxygen species release, thereby compromising their stability and catalytic efficiency. To address these challenges, the SH3-GBD-PDZ (SGP) protein scaffold was applied to assemble a multienzyme system comprising CYP82D26, P450 reductase, and NADP+-dependent aldehyde reductase in desired stoichiometric ratios. Results showed that the coupling efficiency of the P450 system was significantly increased, primarily attributed to the channeling effect of NADPH resulting from the proximity of tethered enzymes and the electrostatic interactions between NADPH and SGP. Assembling this SGP-scaffolded assembly system in Escherichia coli yielded a titer of 240.5 mg/L daidzein with an 86% (2S)-naringenin conversion rate, which showed a 9-fold increase over the free enzymes of the P450 system. These results underscore the potential application of the SGP-scaffolded multienzyme system in enhancing the coupling and catalytic efficiency of the P450 system.

Deciphering the Crucial Roles of the Quorum-Sensing Transcription Factor SdiA in NADPH Metabolism and (S)-Equol Production in Escherichia coli Nissle 1917.

The active metabolite (S)-equol, derived from daidzein by gut microbiota, exhibits superior antioxidative activity compared with its precursor and plays a vital role in human health. As only 25% to 50% of individuals can naturally produce equol when supplied with isoflavone, we engineered probiotic E. coli Nissle 1917 (EcN) to convert dietary isoflavones into (S)-equol, thus offering a strategy to mimic the gut phenotype of natural (S)-equol producers. However, co-fermentation of EcN-eq with fecal bacteria revealed that gut microbial metabolites decreased NADPH levels, hindering (S)-equol production. Transcriptome analysis showed that the quorum-sensing (QS) transcription factor SdiA negatively regulates NADPH levels and (S)-equol biosynthesis in EcN-eq. Screening AHLs showed that SdiA binding to C10-HSL negatively regulates the pentose phosphate pathway, reducing intracellular NADPH levels in EcN-eq. Molecular docking and dynamics simulations investigated the structural disparities in complexes formed by C10-HSL with SdiA from EcN or E. coli K12. Substituting sdiA_EcN in EcN-eq with sdiA_K12 increased the intracellular NADPH/NADP+ ratio, enhancing (S)-equol production by 47%. These findings elucidate the impact of AHL-QS in the gut microbiota on EcN NADPH metabolism, offering insights for developing (S)-equol-producing EcN probiotics tailored to the gut environment.

Developing Ganoderma lucidum as a next-generation cell factory for food and nutraceuticals.

Ganoderma lucidum holds a colossal reservoir of hydrolytic enzymes and therapeutic compounds and can be a sustainable source of proteins and bioactive compounds. Its metabolic versatility, propelled by its rich genome content, provides excellent biosynthetic machinery for innovation-driven pathway engineering. However, robust regulatory networks and low frequency of homologous recombination are critical bottlenecks that limit the development of molecular tools and precise genetic markers for biomanufacturing innovations in this organism. Modern synthetic biology provides tools that could help to accelerate precise multiple gene targeting and editing and untangling the biosynthetic machinery of G. lucidum. This review provides insight into molecular strategies to unwind the regulatory bottlenecks and transform G. lucidum into efficient cell factories for food and nutraceuticals.

Engineering Escherichia coli for cost-effective production of medium-chain fatty acids from soy whey using an optimized galactose-based autoinduction system.

Medium-chain fatty acids (MCFAs) are essential chemical feedstocks. Microbial production of MCFAs offers an attractive alternative to conventional methods, but the costly media and external inducers limit its practical application. To address this issue and make MCFA production more cost-effective, an E.coli platform was developed using soy whey as a medium and galactose as an autoinducer. We first designed an efficient, stringent, homogeneous, and robust galactose-based autoinduction system for the expression of pathway enzymes by rationally engineering the promoter of the galactose-proton symporter (GalP). Subsequently, the intracellular acetyl-CoA availability and NADH regeneration were enhanced to improve the reversal of the ß-oxidation cycle. The resulting strain yielded 8.20 g/L and 16.42 g/L MCFA in pH-controlled batch fermentation and fed-batch fermentation with glucose added using soy whey as medium, respectively. This study provided a cost-effective and promising platform for MCFA production, as well as future strain development for other value-added chemicals production.

Biosynthesis of (S)-Equol from Soy Whey by Metabolically Engineered Escherichia coli.

(S)-Equol is one of the most bioactive metabolites of the isoflavones with immense nutritional and pharmaceutical value. Soy whey is the major liquid byproduct of the soy product processing industries that is rich in nutrients and (S)-equol biosynthetic precursor daidzin. However, it is usually disposed into the sewage, causing high environmental contamination. Herein, we constructed a recombinant Escherichia coli for the biosynthesis of (S)-equol from soy whey. First, we evaluated daidzin-specific transporters and optimized the anaerobically induced Pnar in the (S)-equol biosynthesis cassette to produce (S)-equol from daidzin. Then, sucrase and α-galactosidase were co-expressed to confer sucrose, stachyose, and raffinose utilization capacity on E. coli. Meanwhile, EIIBCAglc was inactivated to eliminate the daidzin transport inhibition induced by glucose. Finally, combining these strategies and optimizing the fermentation conditions, the optimal strain produced 91.5 mg/L of (S)-equol with a yield of 0.96 mol/mol substrates in concentrated soy whey. Overall, this new strategy is an attractive route to broaden the applications of soy whey and achieve the eco-friendly production of (S)-equol.

Recent progress in high-throughput droplet screening and sorting for bioanalysis.

Owing to its ability to isolate single cells and perform high-throughput sorting, droplet sorting has been widely applied in several research fields. Compared with flow cytometry, droplet allows the encapsulation of single cells for cell secretion or lysate analysis. With the rapid development of this technology in the past decade, various droplet sorting devices with high throughput and accuracy have been developed. A droplet sorter with the highest sorting throughput of 30,000 droplets per second was developed in 2015. Since then, increased attention has been paid to expanding the possibilities of droplet sorting technology and strengthening its advantages over flow cytometry. This review aimed to summarize the recent progress in droplet sorting technology from the perspectives of device design, detection signal, actuating force, and applications. Technical details for improving droplet sorting through various approaches are introduced and discussed. Finally, we discuss the current limitations of droplet sorting for single-cell studies along with the existing gap between the laboratory and industry and provide our insights for future development of droplet sorters.

Modular co-culture engineering of Yarrowia lipolytica for amorphadiene biosynthesis.

Amorphadiene is the precursor to synthesize the antimalarial drug artemisinin. The production of amorphadiene and artemisinin from metabolically engineered microbes may provide an alternate to plant secondary metabolite extraction. Microbial consortia can offer division of labor, and microbial co-culture system can be leveraged to achieve cost-efficient production of natural products. Using a co-culture system of Y. lipolytica Po1f and Po1g strains, subcellular localization of ADS gene (encoding amorphadiene synthase) into the endoplasmic reticulum, co-utilization of mixed carbon source, and enlargement of the endoplasmic reticulum (ER) surface area, we were able to significantly improve amorphadiene production in this work. Using Po1g/PPtM and Po1f/AaADSERx3/iGFMPDU strains and co-utilization of 5 µM sodium acetate with 20 g/L glucose in YPD media, amorphadiene titer were increased to 65.094 mg/L. The enlargement of the ER surface area caused by the deletion of the PAH1 gene provided more subcellular ER space for the action of the ADS-tagged gene. It further increased the amorphadiene production to 71.74 mg/L. The results demonstrated that the importance of the spatial localization of critical enzymes, and manipulating metabolic flux in the co-culture of Y. lipolytica can be efficient over a single culture for the bioproduction of isoprenoid-related secondary metabolites in a modular manner.

Sustainable production of genistin from glycerol by constructing and optimizing Escherichia coli.

Genistin is one of the bioactive isoflavone glucosides found in legumes, which have great nutraceutical and pharmaceutical significance. The market available isoflavones are currently produced by direct plant extraction. However, its low abundance in plant and structural complexity hinders access to this phytopharmaceutical via plant extraction or chemical synthesis. Here, the E. coli cell factory for sustainable production of genistin from glycerol was constructed. First, we rebuilt the precursor genistein biosynthesis pathway in E. coli, and its titer was then increased by 668% by identifying rate-limiting steps and applying an artificial protein scaffold system. Then de novo production of genistin from glycerol was achieved by functional screening and introduction of glycosyl-transferases, UDP-glucose pathway and specific genistin efflux pumps, and 48.1 mg/L of genistin was obtained. A further engineered E. coli strain equipped with an improved malonyl-CoA pathway, alternative glycerol-utilization pathways, acetyl-CoA carboxylase (ACC), and CRISPR interference (CRISPRi) mediated regulation produced up to 137.8 mg/L of genistin in shake flask cultures. Finally, 202.7 mg/L genistin was achieved through fed-batch fermentation in a 3-L bioreactor. This study represents the de novo genistin production from glycerol for the first time and will lay the foundation for low-cost microbial production of glucoside isoflavones. In addition, the multiphase workflow may provide a reference for engineering the biosynthetic pathways in other microbial hosts as well, for green manufacturing of complex natural products.

Transcriptomic analysis reveals the antibacterial mechanism of phenolic compounds from kefir fermented soy whey against Escherichia coli 0157:H7 and Listeria monocytogenes.

Transcriptomic analysis was used to investigate the antibacterial mechanism of phenolic compounds from kefir fermented soy whey (FSP) against Escherichia coli 0157:H7 and Listeria monocytogenes. The kefir fermentation increased the concentration of several phenolic aglycones with proven antibacterial efficacy in the FSP. The time-kill curve showed that 2× MICs of the FSP killed >99.9 % of the strains within 2 h of exposure. The checkerboard fractional inhibition concentration (FIC) assay proved that phenolics were the sole antibacterial agent in the FSP. The transmission electron microscope (TEM) photomicrograph corroborated the propidium iodide (PI) uptake, protein, and nucleic acid leakage assays. They demonstrated that the phenolics permeated the cell membrane, disrupted the cytoplasm, and caused cell lysis in the treated cells leading to protein and nucleic acid leakage. The transcriptome analysis revealed that exposure of the cells to MICs of the phenolics induced molecular responses leading to differential expression of 1850 genes in E. coli 0157:H7 and 2090 in L. monocytogenes. The phenolics suppressed the expression of genes crucial for carbohydrate utilization, transmembrane glucose transport, tricarboxylic acid (TCA), and ATP synthesis. The phenolic-induced stress also downregulated the expression of quorum sensing and virulence-related genes, peptidoglycan and phospholipid synthases, and ABC transporters. The cells initiated a resistance response by stimulating the two-component signal transduction systems to trigger the over-expression of a cascade of genes involved in stress resistance, gluconeogenesis, ATPase activity and proton transmembrane transport. Nonetheless, the data indicated that the phenolics suppressed the expression of translational proteins that would have facilitated the resistance and repair of the cell damage caused by the phenolics. The study provides discrete data evidence that FSP could be used to control the pathogenicity and the proliferation of E. coli 0157:H7 and L. monocytogenes in our foods and food systems.

Solid-state fermentation with Rhizopus oligosporus RT-3 enhanced the nutritional properties of soybeans.

Fermented soybean products are favorite foods worldwide because of their nutritional value and health effects. In this study, solid-state fermentation (SSF) of soybeans with Rhizopus oligosporus RT-3 was performed to investigate its nutraceutical potential. A rich enzyme system was released during SSF. Proteins were effectively transformed into small peptides and amino acids. The small peptide content increased by 13.64 times after SSF for 60 h. The antioxidant activity of soybeans was enhanced due to the release of phenolic compounds. The soluble phenolic content increased from 2.55 to 9.28 gallic acid equivalent (GAE) mg/g after SSF for 60 h and exhibited high correlations with microbial enzyme activities during SSF. The potential metabolic pathways being triggered during SSF indicated that the improved nutritional composition of soybean attributed to the biochemical reactions catalyzed by microbial enzymes. These findings demonstrated that SSF could evidently improve the nutritional value and prebiotic potential of soybeans.

Advanced genome-editing technologies enable rapid and large-scale generation of genetic variants for strain engineering and synthetic biology.

Targeted genome editing not only improves our understanding of fundamental rules in life sciences but also affords us versatile toolkits to improve industrially relevant phenotypes in various host cells. In this review, we summarize the recent endeavor to develop efficient genome-editing tools, and emphasize the utility of these tools to generate massive scale of genetic variants. We categorize these tools into traditional recombination-based tools, and more advanced CRISPR as well as RNA-based genome-editing tools. This diverse panel of sophisticated tools has been applied to accelerate strain engineering, upgrade biomanufacturing, and customize biosensing. In parallel with high-throughput phenotyping and AI-based optimization algorithms, we envision that genome-editing technologies will become a driving force to automate and streamline biological engineering, and empower us to address critical challenges in health, environment, energy, and sustainability.

Soybean Whey Bio-Processed Using Weissella hellenica D1501 Protects Neuronal PC12 Cells Against Oxidative Damage.

Soybean whey, as a byproduct of soybean industry, has caused considerable concern recently because of its abundant nutrients. To further utilize soybean whey, it was fermented with Weissella hellenica D1501, and the neuroprotective potency of this beverage was studied in the present work. The phenolic profile and antioxidant capacity of fermented soybean whey (FSBW) were analyzed. The neuroprotective effects were evaluated based on the hydrogen peroxide-stimulated oxidative damage model in a neural-like cell (PC12). Results demonstrated that soybean whey's phenolic contents and antioxidant activities were markedly improved after fermentation. Glycoside isoflavones were efficiently converted into aglycones by W. hellenica D1501. FSBW extract apparently increased cell viability, decreased reactive oxide species levels, and protected antioxidant enzymes in oxidative damage. Furthermore, FSBW effectively reduced apoptosis rate by inhibiting Bax protein and improving Bcl-2 and Bcl-xL proteins. FSBW ameliorated the cell cycle through the decrease of p21 protein and an increase of cyclin A protein. The findings of this study thus suggested that W. hellenica D1501-fermented soybean whey could potentially protect nerve cells against oxidative damage.

Neuroprotective potency of a soy whey fermented by Cordyceps militaris SN-18 against hydrogen peroxide-induced oxidative injury in PC12 cells.

PURPOSE: Soy whey is a byproduct generated from the processing of several soybean products. Its valorization has continued to attract significant research interest in recent times due to the nutritional and bioactive potency of its chemical composition. Herein, the neuroprotective potency of a soy whey fermented by Cordyceps militaris SN-18 against hydrogen peroxide (H2O2)-induced oxidative injury in PC12 cells was investigated. METHODS: The phenolic compositions were analyzed by high-performance liquid chromatography. Antioxidant activities were assessed by ABTS•+ scavenging assay, DPPH radical scavenging assay, reducing power assay, and ferric reducing antioxidant power assay. The neuroprotective effects of fermented soy whey (FSW) were investigated based on the oxidative injury model in PC12 cells. RESULTS: FSW possessed higher total phenolic content and antioxidant activities compared with unfermented soy whey (UFSW) and that most of the isoflavone glycosides were hydrolyzed into their corresponding aglycones during fermentation. The extract from FSW exhibited a greater protective effect on PC12 cells against oxidative injury by promoting cell proliferation, restoring cell morphology, inhibiting lactic dehydrogenase leakage, reducing reactive oxygen species levels, and enhancing antioxidant enzyme activities compared with that from UFSW. Additionally, cell apoptosis was significantly inhibited by FSW through down-regulation of caspase-3, caspase-9, and Bax and up-regulation of Bcl-2 and Bcl-xL. S-phase cell arrest was attenuated by FSW through increasing cyclin A, CDK1 and CDK2, and decreasing p21 protein. CONCLUSION: Fermentation with C. militaris SN-18 could significantly improve the bioactivity of soy whey by enhancing the ability of nerve cells to resist oxidative damage.

Green and efficient in-situ biosynthesis of antioxidant and antibacterial bacterial cellulose using wine pomace.

Biologically active bacterial cellulose (BC) was efficiently synthesized in situ using wine pomace and its hydrolysate. The structural and biomechanical properties together with the biological functions of the BC were investigated. Functional BC from wine pomace and its enzymatic hydrolysate were of high purity and had higher crystallinity indexes (90.61% and 89.88%, respectively) than that from HS medium (82.26%). FTIR results proved the in-situ bindings of polyphenols to the functionalized BC. Compared to BC from HS medium, wine pomace-based BC had more densely packed ultrafine fibrils, higher diameter range distributions of fiber ribbon, but lower thermal decomposition temperatures, as revealed by the SEM micrographs and DSC data. Meanwhile, wine pomace-based BC exhibited higher loads in tensile strength and higher hardness (4.95 ± 0.31 N and 5.13 ± 0.63 N, respectively) than BC in HS medium (3.43 ± 0.14 N). Furthermore, BC synthesized from wine pomace hydrolysate exhibited a slower release rate of phenolic compounds, and possessed more antioxidant activities and better bacteriostatic effects than BC from wine pomace. These results demonstrate that BC synthesized in situ from wine pomace (especially from enzymatic hydrolysate) is a promising biomolecule with a potential application in wound dressing, tissue engineering, and other biomedical fields.

Bio-conversion of kitchen waste into bacterial cellulose using a new multiple carbon utilizing Komagataeibacter rhaeticus: Fermentation profiles and genome-wide analysis.

A cellulose-producing bacterium Komagataeibacter rhaeticus K15 was isolated from kombucha tea, and its metabolic pathways and cellulose synthesis operon were analyzed by genome sequencing. Different from the reported K. rhaeticus, the K15 produced little gluconic acid (2.26 g/L) when glucose was the sole carbon source and has the capacity for high cellulose production (4.76 g/L) with other carbon sources. Furthermore, six nitrogen-fixing genes were found to be responsible for the survival of K15 on a nitrogen-free medium. Based on its fermentation characteristics, K15 was cultured in a kitchen waste medium as a strategy for green and sustainable bacterial cellulose production. The SEM, XRD, and FTIR results indicated that synthesized cellulose has a mean diameter of 40-50 nm nanofiber, good crystallinity, and the same chemical structure. The K15 strain provides a highly viable alternative strategy to reduce the costs of bacterial cellulose production using agro-industrial residues as nutrient sources.

Neuroprotective Potency of Tofu Bio-Processed Using Actinomucor elegans against Hypoxic Injury Induced by Cobalt Chloride in PC12 Cells.

Fermented soybean products have attracted great attention due to their health benefits. In the present study, the hypoxia-injured PC12 cells induced by cobalt chloride (CoCl2) were used to evaluate the neuroprotective potency of tofu fermented by Actinomucor elegans (FT). Results indicated that FT exhibited higher phenolic content and antioxidant activity than tofu. Moreover, most soybean isoflavone glycosides were hydrolyzed into their corresponding aglycones during fermentation. FT demonstrated a significant protective effect on PC12 cells against hypoxic injury by maintaining cell viability, reducing lactic dehydrogenase leakage, and inhibiting oxidative stress. The cell apoptosis was significantly attenuated by the FT through down-regulation of caspase-3, caspases-8, caspase-9, and Bax, and up-regulation of Bcl-2 and Bcl-xL. S-phase cell arrest was significantly inhibited by the FT through increasing cyclin A and decreasing the p21 protein level. Furthermore, treatment with the FT activated autophagy, indicating that autophagy possibly acted as a survival mechanism against CoCl2-induced injury. Overall, FT offered a potential protective effect on nerve cells in vitro against hypoxic damage.

Metabolite dynamics and phytochemistry of a soy whey-based beverage bio-transformed by water kefir consortium.

Integrated metabolomic and metagenomic techniques were used to study the metabolite dynamics and phytochemistry of a soy whey-based beverage bio-transformed by water kefir consortium. The UPLC- MS/MS and HPLC-ESI-MS metabolite quantification and the OPLS-DA result showed that the kefir consortium induced a significant change in the metabolite composition and altered the phytochemistry of the fermented beverage. Bioactive peptide analogues, flavonoids, and glycerophospholipids including N-acetyl-L-phenylalanine, acetyl-DL-leucine; tephcalostan, wogonin, pelargonin, genistein, daidzein, and glycerophosphoserines (PS), glycerophosphoethanolamines (PE) respectively were synthesized while flavonoid glycosides and soyasaponins were degraded in the novel beverage. Furthermore, the beverage showed high ACE inhibitory and DPPH radical scavenging activity of 92.31% and 87.51% respectively. Lactobacillus, Saccharomyces cerevisiae, and Pichia membranifaciens were the predominant microbial groups in the new beverage as revealed by the metagenomic sequence analysis. The study thus provides discrete data evidence that kefir consortium is a viable starter for transforming soy whey into a bioactive beverage.

Heavy metal and microbial safety assessment of raw and cooked pumpkin and Amaranthus viridis leaves grown in Abakaliki, Nigeria.

In this study, heavy metal and microbial safety assessment of raw and cooked pumpkin and Amaranthus viridis leaves grown in Abakaliki, Nigeria, was examined. The levels of lead (Pb), arsenic (As), chromium (Cr), cadmium (Cd), and mercury (Hg) were evaluated using atomic absorption spectrophotometer. The microbial cells were counted and further identified to species level using 16S rDNA and ITS rDNA sequencing analysis at CABI microbial identification services United Kingdom (UK). The results showed that the heavy metal concentrations of lead (10.5, 12.0), arsenic (7.5, 8.5), chromium (0.9, 0.1), and mercury (13.1, 14.0) in the pumpkin and A. viridis leaves, respectively, were above maximum acceptable limit according to relevant national and international food regulatory agencies (Tables 1 and 2). Cooking significantlyreduced the concentrations of the heavy metals at (p > 0.05) to or below, lead (6.8, 8.4), arsenic (5.1, 6.1), chromium (0.6.0.1), and mercury (9.5, 11.4) in the pumpkin and A. viridis leaves, respectively, but still not to international safe limit. The result of the microbial safety assessment showed that the microbial load of both the pumpkin and Amaranthus viridis leaves were above acceptable limit and the contaminating organisms were identified as Escherichia coli (504743), Klebsiella pneumonia (504744b), and Aspergillus flavus (504740).This study therefore shows that the vegetables (pumpkin and A. viridis leaves) contain unacceptable levels of toxic heavy metals and potentially dangerous pathogenic microorganisms, thus present significant health risk for the consumers.

Reclamation of DPK hydrocarbon polluted agricultural soil using a selected bulking agent.

In the present study, laboratory scale bioremediation of dual purpose kerosene (DPK) hydrocarbon polluted soil using bulking agent (saw dust) was carried out. The effect of different parameters such as total petroleum hydrocarbon (TPH), dehydrogenase activity (DHase) and pH on bioremediation performance were evaluated. Studied parameters such as microbial dynamics, percentage degradation (95.20%), DHase (8.20 ± 0.43) were found to be higher in saw dust amended system and significantly differed with control at p < 0.05. Experimental data adequately fitted the first order kinetic thus, generated r(2) values (0.966), first order degradation constant (0.659 d(-1)), and degradation half-life t1/2 = ln2/k (1.05 d). Micrococcus luteus, Bacillus sp., Rhizopus arrhizus and Aspergillus sp. were isolated from the study. The use of saw dust as bulking agent greatly increased biodegradation rate and resulted in effective DPK hydrocarbon clean up. Therefore, saw dust could serve as an effective biostimulant towards improved bioremediation of hydrocarbon polluted environment.