Effects of water-soluble and water-insoluble α-glucans produced in situ by Leuconostoc citreum SH12 on physicochemical properties of fermented soymilk and their structural analysis.

This study investigated the effect of in situ produced water-soluble α-glucan (LcWSG) and water-insoluble α-glucan (LcWIG) from Leuconostoc citreum SH12 on the physicochemical properties of fermented soymilk. α-Glucans produced by Leuc. citreum SH12 improved water-holding capacity, viscosity, viscoelasticity and texture of fermented soymilk. Gtf1365 and Gtf836 of the five putative glucansucrases were responsible for synthesizing LcWSG and LcWIG during soymilk fermentation, respectively. Co-fermentation of soymilk with Gtf1365 and Gtf836 and non-exopolysaccharide-producing Lactiplantibacillus plantarum D1031 indicated that LcWSG effectively hindered the whey separation of fermented soymilk by increasing viscosity, while LcWIG improved hardness, springiness and accelerated protein coagulation. Fermented soymilk gel formation was mainly based on hydrogen bonding and hydrophobic interactions, which were promoted by both LcWSG and LcWIG. LcWIG has a greater effect on α-helix to ß-sheet translation in fermented soymilk, causing more rapid protein aggregation and thicker cross-linked gel network. Structure-based exploration of LcWSG and LcWIG from Leuc. citreum SH12 revealed their distinct roles in the physicochemical properties of fermented soymilk due to their different ratio of α-1,6 and α-1,3 glucosidic linkages and various side chain length. This study may guide the application of the water-soluble and water-insoluble α-glucans in fermented plant protein foods for their quality improvement.

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.

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.

Structural and functional characterization of exopolysaccharide from Leuconostoc citreum BH10 discovered in birch sap.

In this study, Leuconostoc citreum BH10, an endophytic strain, was isolated from aseptically collected xylem sap of birch for the first time, and its exopolysaccharide (LCEPS) production was up to 46.31 g/L in glucan producing medium. The produced LCEPS was purified to obtain two water-soluble fractions, named as LCEPS-1 and LCEPS-2, respectively. The major fraction LCEPS-1 was characterized to be comprised of glucose with average molecular weight of 6.34 × 106 Da. The structure of LCEPS-1 was investigated by spectroscopy analysis, which revealed that LCEPS-1 was identified with containing 90.45 % α-(1,6) linkages in the main chains and 9.55 % α-(1,3) branch linkages. The scanning electron microscope results demonstrated that the dried LCEPS-1 appeared porous surface overlaid with an irregular glittering. The water solubility index (WSI) and water holding capacity (WHC) of LCEPS-1 were 88.02 ± 1.69 % and 241.43 ± 6.38 %, respectively. Besides, it exhibited high thermal stability as well as fine antioxidant activities. Taken together, the results indicated that LCEPS-1 could have good potentiality to be applied in fields of foods, cosmetics, nutraceuticals and pharmaceutical industries as the natural agent.

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.

Tofu by-product soy whey substitutes urea: Reduced ammonia volatilization, enhanced soil fertility and improved fruit quality in cherry tomato production.

Soy whey is an abundant, nutrient-rich and safe wastewater produced in tofu processing, so it is necessary to valorize it instead of discarding it as sewage. Whether soy whey can be used as a fertilizer substitute for agricultural production is unclear. In this study, the effects of soy whey serving as a nitrogen source to substitute urea on soil NH3 volatilization, dissolved organic matter (DOM) components and cherry tomato qualities were investigated by soil column experiment. Results showed that the soil NH4+-N concentrations and pH values of the 50% soy whey fertilizer combined with 50% urea (50%-SW) and 100% soy whey fertilizer (100%-SW) treatments were lower than those of 100% urea treatment (CKU). Compared with CKU, 50%-SW and 100%-SW treatments increased the abundance of ammonia oxidizing bacteria (AOB) by 6.52-100.89%, protease activity by 66.22-83.78%, the contents of total organic carbon (TOC) by 16.97-35.64%, humification index (HIX) of soil DOM by 13.57-17.99%, and average weight per fruit of cherry tomato by 13.46-18.56%, respectively. Moreover, soy whey as liquid organic fertilizer reduced the soil NH3 volatilization by 18.65-25.27% and the fertilization cost by 25.94-51.87% compared with CKU. This study provides a promising option with economic and environmental benefits for soy whey utilization and cherry tomato production, which contributes to the win-win effectiveness of sustainable production for both the soy products industry and agriculture.

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.

Effects of lactic acid bacteria fermented yellow whey on the protein coagulation and isoflavones distribution in soymilk.

This study investigated the soymilk coagulation induced by fermented yellow whey (FYW), which is extensively used as a natural tofu coagulant in China. The aggregations involving proteins and isoflavone particles caused by FYW were analyzed using the proteomic technology and high-performance liquid chromatography, respectively. As indicated, the FYW-induced coagulation of soy proteins mainly occurred at pH 5.80-5.90. When the pH of soymilk decreased, the 7S ß, 11S A3 and some of 11S A1a subunits and SBP, Bd, lectin and TA aggregated the earliest, and later did the 11S A4, other 11S A1a, 11S A2 and 11S A1b subunits. The 7S α and α' subunits and TB showed an obvious delay in aggregation. Moreover, isoflavones in the form of aglycones were more likely to coprecipitate with proteins, compared with glycosides. These results could provide an important reference and assistance for future research on the development of traditional FYW-tofu.

4-{2-[4-(Dimethyl-amino)-phen-yl]ethen-yl}-1-methyl-pyridinium 4-nitro-benzene-sulfonate.

The asymmetric unit of the title salt, C(16)H(19)N(2) (+). C(6)H(4)NO(5)S(-), consists of two cations and two anions. The crystal structure is stabilized by π-π inter-actions between the pyridyl and phenyl rings of the cations, with a centroid-centroid distance of 3.7323 (6) Å.

Is mass-based metabolism rate proportional to surface area in plant leaves? A data re-analysis.

We re-analyzed two large published databases on leaf traits of plant species from seven different biomes, and determined the scaling relationship between leaf metabolism rate (mass-based photosynthesis capacity, A(mass), and mass-based dark respiration, Rd(mass)) and specific leaf area (SLA) across biomes, using a standardized major axis (SMA) method. Overall pooled data produced a scaling exponent of 1.33 for the relationship between A(mass) and SLA, significantly larger than 1.0; and 1.04 between Rd(mass) and SLA. The scaling exponent of the relationship between A(mass) and SLA ranged between 1.23 (in tropical forest) and 1.66 (in alpine biome), and it was significantly larger in alpine (1.66) and grass/meadow (1.52) biomes than in tropical forest (1.23) and wetland (1.27). The exponent of the relationship between Rd(mass) and SLA, however, was much smaller in wetland (1.05) than in temperate forest (1.29) and tropical rainforest (1.65). In general, the predicated universal scaling relationship that the mass-based metabolism rate should be proportional to surface area in organisms is not applicable at the leaf-level in plants. Rather, the large slope difference of the relationship between leaf metabolism rate and SLA found among biomes indicates that the strength of the selective forces driving the scaling relationship is different among the biomes. The result basically suggests the importance of increasing SLA to plant carbon gain in stressful environments and to carbon loss in favorable habitats, and therefore has an important implication for survival strategies of plants in different biomes.