Understanding D-xylonic acid accumulation: a cornerstone for better metabolic engineering approaches.

The xylose oxidative pathway (XOP) has been engineered in microorganisms for the production of a wide range of industrially relevant compounds. However, the performance of metabolically engineered XOP-utilizing microorganisms is typically hindered by D-xylonic acid accumulation. It acidifies the media and perturbs cell growth due to toxicity, thus curtailing enzymatic activity and target product formation. Fortunately, from the growing portfolio of genetic tools, several strategies that can be adapted for the generation of efficient microbial cell factories have been implemented to address D-xylonic acid accumulation. This review centers its discussion on the causes of D-xylonic acid accumulation and how to address it through different engineering and synthetic biology techniques with emphasis given on bacterial strains. In the first part of this review, the ability of certain microorganisms to produce and tolerate D-xylonic acid is also tackled as an important aspect in developing efficient microbial cell factories. Overall, this review could shed some insights and clarity to those working on XOP in bacteria and its engineering for the development of industrially applicable product-specialist strains. KEY POINTS: D-Xylonic acid accumulation is attributed to the overexpression of xylose dehydrogenase concomitant with basal or inefficient expression of enzymes involved in D-xylonic acid assimilation. Redox imbalance and insufficient cofactors contribute to D-xylonic acid accumulation. Overcoming D-xylonic acid accumulation can increase product formation among engineered strains. Engineering strategies involving enzyme engineering, evolutionary engineering, coutilization of different sugar substrates, and synergy of different pathways could potentially address D-xylonic acid accumulation.

Enhanced glycolic acid yield through xylose and cellobiose utilization by metabolically engineered Escherichia coli.

Microbial biorefinery is a promising route toward sustainable production of glycolic acid (GA), a valuable raw material for various industries. However, inherent microbial GA production has limited substrate consumption using either D-xylose or D-glucose as carbon catabolite repression (CCR) averts their co-utilization. To bypass CCR, a GA-producing strain using D-xylose via Dahms pathway was engineered to allow cellobiose uptake. Unlike glucose, cellobiose was assimilated and intracellularly degraded without repressing D-xylose uptake. The final GA-producing E. coli strain (CLGA8) has an overexpressed cellobiose phosphorylase (cep94A) from Saccharophagus degradans 2-40 and an activated glyoxylate shunt pathway. Expression of cep94A improved GA production reaching the maximum theoretical yield (0.51 g GA g-1 xylose), whereas activation of glyoxylate shunt pathway enabled GA production from cellobiose, which further increased the GA titer (2.25 g GA L-1). To date, this is the highest reported GA yield from D-xylose through Dahms pathway in an engineered E. coli with cellobiose as co-substrate.

Engineering of xylose metabolism in Escherichia coli for the production of valuable compounds.

The lignocellulosic sugar d-xylose has recently gained prominence as an inexpensive alternative substrate for the production of value-added compounds using genetically modified organisms. Among the prokaryotes, Escherichia coli has become the de facto host for the development of engineered microbial cell factories. The favored status of E. coli resulted from a century of scientific explorations leading to a deep understanding of its systems. However, there are limited literature reviews that discuss engineered E. coli as a platform for the conversion of d-xylose to any target compounds. Additionally, available critical review articles tend to focus on products rather than the host itself. This review aims to provide relevant and current information about significant advances in the metabolic engineering of d-xylose metabolism in E. coli. This focusses on unconventional and synthetic d-xylose metabolic pathways as several review articles have already discussed the engineering of native d-xylose metabolism. This paper, in particular, is essential to those who are working on engineering of d-xylose metabolism using E. coli as the host.

Overexpression and characterization of a novel GH16 ß-agarase (Aga1) from Cellulophaga omnivescoria W5C.

OBJECTIVE: To identify and characterize a new ß-agarase from Cellulophaga omnivescoria W5C capable of producing biologically-active neoagarooligosaccharides from agar. RESULTS: The ß-agarase, Aga1, has signal peptides on both N- and C-terminals, which are involved in the type IX secretion system. It shares 75% protein sequence identity with AgaD from Zobellia galactanivorans and has a molecular weight of 54 kDa. Biochemical characterization reveals optimum agarolytic activities at pH 7-8 and temperature 30-45 °C. Aga1 retains at least 33% activity at temperatures lower than the sol-gel transition state of agarose. Metal ions are generally not essential, but calcium and potassium enhance its activity whereas iron and zinc are inhibitory. Finally, hydrolysis of agarose with Aga1 yields neoagarotetraose, neoagarohexaose, and neoagarooctaose. CONCLUSIONS: Aga1 displays unique traits such as moderate psychrophilicity, stability, and synergy with other agarases, which makes it an excellent candidate for biosynthetic production of neoagarooligosaccharides from agar.

A pH-responsive genetic sensor for the dynamic regulation of D-xylonic acid accumulation in Escherichia coli.

The xylose oxidative pathway (XOP) is continuously gaining prominence as an alternative for the traditional pentose assimilative pathways in prokaryotes. It begins with the oxidation of D-xylose to D-xylonic acid, which is further converted to α-ketoglutarate or pyruvate + glycolaldehyde through a series of enzyme reactions. The persistent drawback of XOP is the accumulation of D-xylonic acid intermediate that causes culture media acidification. This study addresses this issue through the development of a novel pH-responsive synthetic genetic controller that uses a modified transmembrane transcription factor called CadCΔ. This genetic circuit was tested for its ability to detect extracellular pH and to control the buildup of D-xylonic acid in the culture media. Results showed that the pH-responsive genetic sensor confers dynamic regulation of D-xylonic acid accumulation, which adjusts with the perturbation of culture media pH. This is the first report demonstrating the use of a pH-responsive transmembrane transcription factor as a transducer in a synthetic genetic circuit that was designed for XOP. This may serve as a benchmark for the development of other genetic controllers for similar pathways that involve acidic intermediates.

Correction to: A pH-responsive genetic sensor for the dynamic regulation of D-xylonic acid accumulation in Escherichia coli.

In the published version, the y-axis data of Fig. 3c was incorrectly inserted (OD600 instead of D-xylonate (g L-1) and the x-axes of Figs. 3b, 3d, 3e and 3f ended at 48 h instead of 72 h. See the correct Fig. 3 below.

Discovering a novel D-xylonate-responsive promoter: the PyjhI-driven genetic switch towards better 1,2,4-butanetriol production.

The capability of Escherichia coli to catabolize D-xylonate is a crucial component for building and optimizing the Dahms pathway. It relies on the inherent dehydratase and keto-acid aldolase activities of E. coli. Although the biochemical characteristics of these enzymes are known, their inherent expression regulation remains unclear. This knowledge is vital for the optimization of D-xylonate assimilation, especially in addressing the problem of D-xylonate accumulation, which hampers both cell growth and target product formation. In this report, molecular biology techniques and synthetic biology tools were combined to build a simple genetic switch controller for D-xylonate. First, quantitative and relative expression analysis of the gene clusters involved in D-xylonate catabolism were performed, revealing two D-xylonate-inducible operons, yagEF and yjhIHG. The 5'-flanking DNA sequence of these operons were then subjected to reporter gene assays which showed PyjhI to have low background activity and wide response range to D-xylonate. A PyjhI-driven synthetic genetic switch was then constructed containing feedback control to autoregulate D-xylonate accumulation and to activate the expression of the genes for 1,2,4-butanetriol (BTO) production. The genetic switch effectively reduced D-xylonate accumulation, which led to 31% BTO molar yield, the highest for direct microbial fermentation systems thus far. This genetic switch can be further modified and employed in the production of other compounds from D-xylose through the xylose oxidative pathway.

Improved cell growth and biosynthesis of glycolic acid by overexpression of membrane-bound pyridine nucleotide transhydrogenase.

The non-conventional D-xylose metabolism called the Dahms pathway which only requires the expression of at least three enzymes to produce pyruvate and glycolaldehyde has been previously engineered in Escherichia coli. Strains that rely on this pathway exhibit lower growth rates which were initially attributed to the perturbed redox homeostasis as evidenced by the lower intracellular NADPH concentrations during exponential growth phase. NADPH-regenerating systems were then tested to restore the redox homeostasis. The membrane-bound pyridine nucleotide transhydrogenase, PntAB, was overexpressed and resulted to a significant increase in biomass and glycolic acid titer and yield. Furthermore, expression of PntAB in an optimized glycolic acid-producing strain improved the growth and product titer significantly. This work demonstrated that compensating for the NADPH demand can be achieved by overexpression of PntAB in E. coli strains assimilating D-xylose through the Dahms pathway. Consequently, increase in biomass accumulation and product concentration was also observed.

Everyone loves an underdog: metabolic engineering of the xylose oxidative pathway in recombinant microorganisms.

The D-xylose oxidative pathway (XOP) has recently been employed in several recombinant microorganisms for growth or for the production of several valuable compounds. The XOP is initiated by D-xylose oxidation to D-xylonolactone, which is then hydrolyzed into D-xylonic acid. D-Xylonic acid is then dehydrated to form 2-keto-3-deoxy-D-xylonic acid, which may be further dehydrated then oxidized into α-ketoglutarate or undergo aldol cleavage to form pyruvate and glycolaldehyde. This review introduces a brief discussion about XOP and its discovery in bacteria and archaea, such as Caulobacter crescentus and Haloferax volcanii. Furthermore, the current advances in the metabolic engineering of recombinant strains employing the XOP are discussed. This includes utilization of XOP for the production of diols, triols, and short-chain organic acids in Escherichia coli, Saccharomyces cerevisiae, and Corynebacterium glutamicum. Improving the D-xylose uptake, growth yields, and product titer through several metabolic engineering techniques bring some of these recombinant strains close to industrial viability. However, more developments are still needed to optimize the XOP pathway in the host strains, particularly in the minimization of by-product formation.

Draft Genome Sequence of Newly Isolated Agarolytic Bacteria Cellulophaga omnivescoria sp. nov. W5C Carrying Several Gene Loci for Marine Polysaccharide Degradation.

The continued research in the isolation of novel bacterial strains is inspired by the fact that native microorganisms possess certain desired phenotypes necessary for recombinant microorganisms in the biotech industry. Most studies have focused on the isolation and characterization of strains from marine ecosystems as they present a higher microbial diversity than other sources. In this study, a marine bacterium, W5C, was isolated from red seaweed collected from Yeosu, South Korea. The isolate can utilize several natural polysaccharides such as agar, alginate, carrageenan, and chitin. Genome sequence and comparative genomics analyses suggest that strain W5C belongs to a novel species of the Cellulophaga genus, from which the name Cellulophaga omnivescoria sp. nov. is proposed. Its genome harbors 3,083 coding sequences and 146 carbohydrate-active enzymes (CAZymes). Compared to other reported Cellulophaga species, the genome of W5C contained a higher proportion of CAZymes (4.7%). Polysaccharide utilization loci (PUL) for agar, alginate, and carrageenan were identified in the genome, along with other several putative PULs. These PULs are excellent sources for discovering novel hydrolytic enzymes and pathways with unique characteristics required for biorefinery applications, particularly in the utilization of marine renewable biomass. The type strain is JCM 32108T (= KCTC 13157BPT).

Engineering Escherichia coli for glycolic acid production from D-xylose through the Dahms pathway and glyoxylate bypass.

Glycolic acid (GA) is an ⍺-hydroxy acid used in cosmetics, packaging, and medical industries due to its excellent properties, especially in its polymeric form. In this study, Escherichia coli was engineered to produce GA from D-xylose by linking the Dahms pathway, the glyoxylate bypass, and the partial reverse glyoxylate pathway (RGP). Initially, a GA-producing strain was constructed by disrupting the xylAB and glcD genes in the E. coli genome and overexpressing the xdh(Cc) from Caulobacter crescentus. This strain was further improved through modular optimization of the Dahms pathway and the glyoxylate bypass. Results for module 1 showed that the rate-limiting step of the Dahms pathway was the xylonate dehydratase reaction, and the overexpression of yagF was sufficient to overcome this bottleneck. Furthermore, the appropriate aldolase gene for module 1 was proven to be yagE. The results also show that overexpression of the lactaldehyde dehydrogenase gene, aldA, is needed to increase the GA production while the overexpression of glyoxylate reductase gene, ycdW, was only essential when the glyoxylate bypass was active. On the other hand, the module 2 enzymes AceA and AceK were vital in activating the glyoxylate bypass, while the RGP enzymes were dispensable. The final strain (GA19) produced 4.57 g/L GA with a yield of 0.46 g/g from D-xylose. So far, this is the highest value achieved for GA production in engineered E. coli through the Dahms pathway.

Identification and characterization of a thermostable endolytic ß-agarase Aga2 from a newly isolated marine agarolytic bacteria Cellulophaga omnivescoria W5C.

Research on the enzymatic breakdown of seaweed-derived agar has recently gained attention due to the progress in green technologies for marine biomass utilization. The enzymes known as agarases catalyze the cleavage of glycosidic bonds within the polysaccharide. In this study, a new ß-agarase, Aga2, was identified from Cellulophaga omnivescoria W5C. Aga2 is one of four putative agarases from the W5C genome, and it belongs to the glycoside hydrolase 16 family. It was shown to be exclusive to the Cellulophaga genus. Agarase activity assays showed that Aga2 is an endolytic-type ß-agarase that produces tetrameric and hexameric neoagaro-oligosaccharides, with optimum activity at 45°C and pH 8.0. Zinc ions slightly enhanced its activity while manganese ions had inhibitory effects even at very low concentrations. Aga2 has a Km of 2.59mgmL-1 and Vmax of 275.48Umg-1. The Kcat is 1.73×102s-1, while the Kcat/Km is 8.04×106s-1M-1. Aga2 also showed good thermostability at 45°C and above, and retained >90% of its activity after repeated freeze-thaw cycles. Bioinformatic analysis of its amino acid sequence revealed that intrinsic properties of the protein (e.g. presence of certain dipeptides and the relative volume occupied by aliphatic amino acids) and tertiary structural elements (e.g. presence of salt bridges, hydrophobic interactions and H-bonding) contributed to its thermostability.

Performance evaluation of poly-urethane foam packed-bed chemical scrubber for the oxidative absorption of NH3 and H2S gases.

The feasibility of open-pore polyurethane (PU) foam as packing material for wet chemical scrubber was tested for NH3 and H2S removals. The foam is inexpensive, light-weight, highly porous (low pressure drop) and provides large surface area per unit volume, which are desirable properties for enhanced gas/liquid mass transfer. Conventional HCl/HOCl (for NH3) and NaOH/NaOCl (for H2S) scrubbing solutions were used to absorb and oxidize the gases. Assessment of the wet chemical scrubbers reveals that pH and ORP levels are important to maintain the gas removal efficiencies >95%. A higher re-circulation rate of scrubbing solutions also proved to enhance the performance of the NH3 and H2S columns. Accumulation of salts was confirmed by the gradual increase in total dissolved solids and conductivity values of scrubbing solutions. The critical elimination capacities at >95% gas removals were found to be 5.24 g NH3-N/m3-h and 17.2 g H2S-S/m3-h at an empty bed gas residence time of 23.6 s. Negligible pressure drops (< 4 mm H2O) after continuous operation demonstrate the suitability of PU as a practical packing material in wet chemical scrubbers for NH3 and H2S removals from high-volume dilute emissions.

Enhanced yield of ethylene glycol production from d-xylose by pathway optimization in Escherichia coli.

The microbial production of renewable ethylene glycol (EG) has been gaining attention recently due to its growing importance in chemical and polymer industries. EG has been successfully produced biosynthetically from d-xylose through several novel pathways. The first report on EG biosynthesis employed the Dahms pathway in Escherichia coli wherein 71% of the theoretical yield was achieved. This report further improved the EG yield by implementing metabolic engineering strategies. First, d-xylonic acid accumulation was reduced by employing a weak promoter which provided a tighter control over Xdh expression. Second, EG yield was further improved by expressing the YjgB, which was identified as the most suitable aldehyde reductase endogenous to E. coli. Finally, cellular growth, d-xylose consumption, and EG yield were further increased by blocking a competing reaction. The final strain (WTXB) was able to reach up to 98% of the theoretical yield (25% higher as compared to the first study), the highest reported value for EG production from d-xylose.

SH003 selectively induces p73­dependent apoptosis in triple­negative breast cancer cells.

Triple-negative breast cancer (TNBC) is a breast cancer subtype that has an aggressive phenotype, is highly metastatic, has limited treatment options and is associated with a poor prognosis. In addition, metastatic TNBC has no preferred standard chemotherapy due to resistance to anthracyclines and taxanes. The present study demonstrated that a herbal extract, SH003, reduced cell viability and induced apoptosis in TNBC without cell cytotoxicity. Cell viability was examined using trypan blue exclusion and colony formation assays, which revealed a decrease in the cell viability. Additionally, apoptosis was determined using flow cytometry and a sub­G1 assay, which revealed an increase in the proportion of cells in the sub­G1 phase. The present study investigated the anticancer effect of SH003 in the Hs578T, MDA­MB­231 and ZR­751 TNBC cell lines, and in the MCF7 and T47D non­TNBC cell lines. Western blot analysis revealed that the expression levels of poly­ADP­ribose polymerase (PARP) cleavage protein in cells treated with SH003 were increased dose­dependent manner, indicating that SH003 induced apoptosis via a caspase­dependent pathway. Pre­treatment with the caspase inhibitor Z­VAD reduced SH003­induced apoptosis was examined using trypan blue exclusion. Moreover, SH003 treatment enhanced the p73 levels in MDA­MB­231 cells but not in MCF7 cells. Transfection of p73 small interfering RNA (siRNA) in MDA­MB0231 cells revealed that the apoptotic cell death induced by SH003 was significantly impaired in comparison with scramble siRNA transfected MDA­MB­231 cells. This was examined using trypan blue exclusion and flow cytometry analysis (sub­G1). In addition, SH003 and paclitaxel exhibited synergistic anticancer effects on TNBC cells. The results indicate that SH003 exerts its anticancer effect via p73 protein induction and exhibits synergistic anticancer effects when combined with paclitaxel.

Overexpression and secretion of AgaA7 from Pseudoalteromonas hodoensis sp. nov in Bacillus subtilis for the depolymerization of agarose.

Interest in agar or agarose-based pharmaceutical products has driven the search for potent agarolytic enzymes. An extracellular ß-agarase (AgaA7) recently isolated from Pseudoalteromonas hodoensis sp. nov was expressed in Bacillus subtilis, which was chosen due to its capability to overproduce and secrete functional enzymes. Phenotypic analysis showed that the engineered B. subtilis secreted a functional AgaA7 when fused with the aprE signal peptide (SP) at the amino-terminus. The maximum agarolytic activity was observed during the late logarithmic phase. To further improve the secretion of AgaA7, an expression library of AgaA7 fused to different naturally occurring B. subtilis SPs was created. The amount of AgaA7 secreted by the clones was compared through activity assay, immuno-blot, and purification via affinity chromatography. Although the aprE SP can readily facilitate the secretion of AgaA7, other SPs such as yqgA, pel, and lipA were relatively more efficient. Among these SPs, lipA was the most efficient in improving the secretion of AgaA7.The use of B. subtilis as host for the expression and secretion of agarolytic and other hydrolytic enzymes can be a useful tool in the field of white biotechnology.

Combined treatment with vitamin C and sulindac synergistically induces p53- and ROS-dependent apoptosis in human colon cancer cells.

Sulindac has anti-neoplastic properties against colorectal cancers; however, its use as a chemopreventive agent has been limited due to toxicity and efficacy concerns. Combinatorial treatment of colorectal cancers has been attempted to maximize anti-cancer efficacy with minimal side effects by administrating NSAIDs in combination with other inhibitory compounds or drugs such as l-ascorbic acid (vitamin C), which is known to exhibit cytotoxicity towards various cancer cells at high concentrations. In this study, we evaluated a combinatorial strategy utilizing sulindac and vitamin C. The death of HCT116 cells upon combination therapy occurred via a p53-mediated mechanism. The combination therapeutic resistance developed in isogenic p53 null HCT116 cells and siRNA-mediated p53 knockdown HCT116 cells, but the exogenous expression of p53 in p53 null isogenic cells resulted in the induction of cell death. In addition, we investigated an increased level of intracellular ROS (reactive oxygen species), which was preceded by p53 activation. The expression level of PUMA (p53-upregulated modulator of apoptosis), but not Bim, was significantly increased in HCT116 cells in response to the combination treatment. Taken together, our results demonstrate that combination therapy with sulindac and vitamin C could be a novel anti-cancer therapeutic strategy for p53 wild type colon cancers.

SAHA, an HDAC inhibitor, overcomes erlotinib resistance in human pancreatic cancer cells by modulating E-cadherin.

Pancreatic cancer is one of the most lethal cancers and remains a major unsolved health problem. Less than 20 % of patients are surgical candidates, and the median survival for non-resected patients is approximately 3 to 4 months. Despite the existence of many conventional cancer therapies, few targeted therapies have been developed for pancreatic cancer. Combination therapy using erlotinib and gemcitabine is an approved standard chemotherapy for advanced pancreatic cancer, but it has marginal therapeutic benefit. To try to improve the therapeutic outlook, we studied the efficacy of another combination treatment and the relevance to E-cadherin in human pancreatic cancer cells. We treated two human pancreatic cancer cell lines with the histone deacetylase inhibitor (HDACi) SAHA. Interestingly, in these Panc-1 and Capan1 cells, we observed that the expression levels of E-cadherin and phosphorylated EGFR were gradually upregulated after treatment with SAHA. Furthermore, these cells underwent induced cell death after exposure to the combination treatment of SAHA and erlotinib. In Panc-1 cells, overexpression of E-cadherin activated the phosphorylation of EGFR and increased the cell sensitivity to erlotinib. In Capan1 cells, knocking down E-cadherin decreased the expression of phosphorylated EGFR, and these cells did not respond to erlotinib. Therefore, we demonstrated the efficacy of the combined treatment with SAHA and erlotinib in human pancreatic cancer cells, and we determined that the increased efficacy was due, at least in part, to the effects of SAHA on the expression of E-cadherin. Our studies suggest that E-cadherin may be a potent biomarker for pancreatic cancer.

Identification of aldehyde reductase catalyzing the terminal step for conversion of xylose to butanetriol in engineered Escherichia coli.

Biosynthetic pathways for the production of biofuels often rely on inherent aldehyde reductases (ALRs) of the microbial host. These native ALRs play vital roles in the success of the microbial production of 1,3-propanediol, 1,4-butanediol, and isobutanol. In the present study, the main ALR for 1,2,4-butanetriol (BT) production in Escherichia coli was identified. Results of real-time PCR analysis for ALRs in EWBT305 revealed the increased expression of adhP, fucO, adhE, and yqhD genes during BT production. The highest increase of expression was observed up to four times in yqhD. Singular deletion of adhP, fucO, or adhE gene showed marginal differences in BT production compared to that of the parent strain, EWBT305. Remarkably, yqhD gene deletion (KBTA4 strain) almost completely abolished BT production while its re-introduction (wild-type gene with its native promoter) on a low copy plasmid restored 75 % of BT production (KBTA4-2 strain). This suggests that yqhD gene is the main ALR of the BT pathway. In addition, KBTA4 showed almost no NADPH-dependent ALR activity, but was also restored upon re-introduction of the yqhD gene (KBTA4-2 strain). Therefore, the required ALR activity to complete the BT pathway was mainly contributed by YqhD. Increased gene expression and promiscuity of YqhD were both found essential factors to render YqhD as the key ALR for the BT pathway.

Monascus pilosus-fermented black soybean inhibits lipid accumulation in adipocytes and in high-fat diet-induced obese mice.

OBJECTIVE: To explore the anti-obesity effects and the mechanism of action of Monascus pilosus(M. pilosus)-fermented black soybean (MFBS) extracts (MFBSE) and MFBS powders (MFBSP) in adipocytes and high-fat diet (HFD)-induced obese mice, respectively. METHODS: Black soybean was fermented with M. pilosus, and the main constituents in MFBS were analyzed by HPLC analysis. In vitro, MFBSE were examined for anti-adipogenic effects using Oil-Red O staining. In vivo, mice were fed a normal-fat diet (NFD) control, HFD control or HFD containing 1 g/kg MFBSP for 12 weeks, and then body weight gain and tissues weight measured. Real-time PCR and western blot assay were used to determine the mechanism of anti-adipogenic effects. RESULTS: MFBSE inhibited lipid accumulation in 3T3-L1 adipocytes without exerting cell cytotoxicity. MFBSP treatment in HFD-fed mice significantly decreased the body weight gain compared with the HFD control mice. MFBSE and MFBSP treatment resulted in significantly lower mRNA levels of adipogenesis-related genes, such as peroxisome proliferator-activated receptor γ(PPAR γ), fatty acid-binding protein 4 (FABP4), and fatty acid synthase (FAS), in adipocytes and in white adipose tissue (WAT) of HFD-induced obese mice. CONCLUSIONS: These results suggest that the anti-obesity effects of MFBS are elicited by regulating the expression of adipogenesis-related genes in adipocytes and WAT of HFD-induced obese mice.