Identification of FtfL as a novel target of berberine in intestinal bacteria.

BACKGROUND: Berberine (BBR) is a commonly used anti-intestinal inflammation drug, and its anti-cancer activity has been found recently. BBR can intervene and control malignant colorectal cancer (CRC) through intestinal microbes, but the direct molecular target and related mechanism are unclear. This study aimed to identify the target of BBR and dissect related mechanisms against the occurrence and development of CRC from the perspective of intestinal microorganisms. RESULTS: Here, we found that BBR inhibits the growth of several CRC-driving bacteria, especially Peptostreptococcus anaerobius. By using a biotin-conjugated BBR derivative, we identified the protein FtfL (formate tetrahydrofolate ligase), a key enzyme in C1 metabolism, is the molecular target of BBR in P. anaerobius. BBR exhibits strong binding affinity and potent inhibition on FtfL. Based on this, we determined the crystal structure of PaFtfL (P. anaerobius FtfL)-BBR complex and found that BBR can not only interfere with the conformational flexibility of PaFtfL tetramer by wedging the tetramer interface but also compete with its substrate ATP for binding within the active center. In addition, the enzymatic activities of FtfL homologous proteins in human tumor cells can also be inhibited by BBR. CONCLUSIONS: In summary, our study has identified FtfL as a direct target of BBR and uncovered molecular mechanisms involved in the anti-CRC of BBR. BBR interferes with intestinal pathogenic bacteria by targeting FtfLs, suggesting a new means for controlling the occurrence and development of CRC.

Developing an endogenous quorum-sensing based CRISPRi circuit for autonomous and tunable dynamic regulation of multiple targets in Streptomyces.

Quorum-sensing (QS) mediated dynamic regulation has emerged as an effective strategy for optimizing product titers in microbes. However, these QS-based circuits are often created on heterologous systems and require careful tuning via a tedious testing/optimization process. This hampers their application in industrial microbes. Here, we design a novel QS circuit by directly integrating an endogenous QS system with CRISPRi (named EQCi) in the industrial rapamycin-producing strain Streptomyces rapamycinicus. EQCi combines the advantages of both the QS system and CRISPRi to enable tunable, autonomous, and dynamic regulation of multiple targets simultaneously. Using EQCi, we separately downregulate three key nodes in essential pathways to divert metabolic flux towards rapamycin biosynthesis and significantly increase its titers. Further application of EQCi to simultaneously regulate these three key nodes with fine-tuned repression strength boosts the rapamycin titer by ∼660%, achieving the highest reported titer (1836 ± 191 mg/l). Notably, compared to static engineering strategies, which result in growth arrest and suboptimal rapamycin titers, EQCi-based regulation substantially promotes rapamycin titers without affecting cell growth, indicating that it can achieve a trade-off between essential pathways and product synthesis. Collectively, this study provides a convenient and effective strategy for strain improvement and shows potential for application in other industrial microorganisms.

Molecular mechanism of environmental d-xylose perception by a XylFII-LytS complex in bacteria.

d-xylose, the main building block of plant biomass, is a pentose sugar that can be used by bacteria as a carbon source for bio-based fuel and chemical production through fermentation. In bacteria, the first step for d-xylose metabolism is signal perception at the membrane. We previously identified a three-component system in Firmicutes bacteria comprising a membrane-associated sensor protein (XylFII), a transmembrane histidine kinase (LytS) for periplasmic d-xylose sensing, and a cytoplasmic response regulator (YesN) that activates the transcription of the target ABC transporter xylFGH genes to promote the uptake of d-xylose. The molecular mechanism underlying signal perception and integration of these processes remains elusive, however. Here we purified the N-terminal periplasmic domain of LytS (LytSN) in a complex with XylFII and determined the conformational structures of the complex in its d-xylose-free and d-xylose-bound forms. LytSN contains a four-helix bundle, and XylFII contains two Rossmann fold-like globular domains with a xylose-binding cleft between them. In the absence of d-xylose, LytSN and XylFII formed a heterodimer. Specific binding of d-xylose to the cleft of XylFII induced a large conformational change that closed the cleft and brought the globular domains closer together. This conformational change led to the formation of an active XylFII-LytSN heterotetramer. Mutations at the d-xylose binding site and the heterotetramer interface diminished heterotetramer formation and impaired the d-xylose-sensing function of XylFII-LytS. Based on these data, we propose a working model of XylFII-LytS that provides a molecular basis for d-xylose utilization and metabolic modification in bacteria.

Improving the performance of solventogenic clostridia by reinforcing the biotin synthetic pathway.

An efficient production process is important for industrial microorganisms. The cellular efficiency of solventogenic clostridia, a group of anaerobes capable of producing a wealth of bulk chemicals and biofuels, must be improved for competitive commercialization. Here, using Clostridium acetobutylicum, a species of solventogenic clostridia, we revealed that the insufficient biosynthesis of biotin, a pivotal coenzyme for many important biological processes, is a major limiting bottleneck in this anaerobe's performance. To address this problem, we strengthened the biotin synthesis of C. acetobutylicum by overexpressing four relevant genes involved in biotin transport and biosynthesis. This strategy led to faster growth and improved the titer and productivity of acetone, butanol and ethanol (ABE solvents) of C. acetobutylicum in both biotin-containing and biotin-free media. Expressionally modulating these four genes by modifying the ribosome binding site further promoted cellular performance, achieving ABE solvent titer and productivity as high as 21.9g/L and 0.30g/L/h, respectively, in biotin-free medium; these values exceeded those of the wild-type strain by over 30%. More importantly, biotin synthesis reinforcement also conferred improved ability of C. acetobutylicum to use hexose and pentose sugars, further demonstrating the potential of this metabolic-engineering strategy in solventogenic clostridia.

A toolbox for genetic manipulation in intestinal Clostridium symbiosum.

Gut microbes are closely related with human health, but remain much to learn. Clostridium symbiosum is a conditionally pathogenic human gut bacterium and regarded as a potential biomarker for early diagnosis of intestinal tumors. However, the absence of an efficient toolbox that allows diverse genetic manipulations of this bacterium limits its in-depth studies. Here, we obtained the complete genome sequence of C. symbiosum ATCC 14940, a representative strain of C. symbiosum. On this basis, we further developed a series of genetic manipulation methods for this bacterium. Firstly, following the identification of a functional replicon pBP1 in C. symbiosum ATCC 14940, a highly efficient conjugative DNA transfer method was established, enabling the rapid introduction of exogenous plasmids into cells. Next, we constructed a dual-plasmid CRISPR/Cas12a system for genome editing in this bacterium, reaching over 60 % repression for most of the chosen genes as well as efficient deletion (>90 %) of three target genes. Finally, this toolbox was used for the identification of crucial functional genes, involving growth, synthesis of important metabolites, and virulence of C. symbiosum ATCC 14940. Our work has effectively established and optimized genome editing methods in intestinal C. symbiosum, thereby providing strong support for further basic and application research in this bacterium.

Expression of yes-associated protein, ß-catenin and smoothened, and their clinical significance in invasive breast cancer.

The expression profile and role of yes-associated protein (YAP) in occurrence and development of breast cancer is ambiguous. The present study aimed to explore the relationship among the YAP, ß-catenin and smoothened (SMO) signaling pathways to provide a theoretical basis for the clinical diagnosis and treatment of invasive breast cancer. Immunohistochemistry was used to determine the protein expression levels of YAP, ß-catenin and SMO in tumor, tumor-adjacent and normal breast tissue. The possible association between the expression levels of these three proteins and the clinicopathological features of patients with breast cancer was then analyzed by the χ2 test. The protein expression of YAP was found to be downregulated, whilst ß-catenin and SMO expression were found to be upregulated in tumor tissues as compared with that in normal breast tissues. In addition, the expression of YAP in breast cancer tissues was found to be associated with that of human epidermal growth factor receptor 2 (HER2), progesterone and estrogen receptors. By contrast, the protein expression of ß-catenin and SMO in breast cancer tissues was only associated with HER2. There was a negative correlation between the expression of YAP and SMO protein in breast cancer tissues. Compared with that in the changes in each of YAP, ß-catenin and SMO protein expression levels individually, their combined changes in expression were demonstrated to associate significantly with the tumor histological grade. To conclude, data from the present study suggest that the combined protein expression of YAP, ß-catenin and SMO can be used as a prognostic indicator for the treatment of invasive breast cancer.

Novel peripheral blood parameters as predictors of neoadjuvant chemotherapy response in breast cancer.

The neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), systemic immune severity index (SII), and prognostic nutritional index (PNI) are associated with the prognosis of gastric, lung, and breast cancers. However, the predictive value of pathological complete response (pCR) rates in patients with breast cancer treated with neoadjuvant chemotherapy (NAC) remains unclear. This retrospective study explored the correlation between each index and the efficacy of neoadjuvant chemotherapy in patients with breast cancer and assessed the relationship between changes before and after neoadjuvant chemotherapy. We enrolled 95 patients with locally advanced breast cancer who received neoadjuvant therapy for breast cancer at the Second Affiliated Hospital of Fujian Medical University from April 2020 to April 2022. Based on postoperative pathology, patients were divided into pCR and non-pCR groups. Between-group differences and efficacy prediction ability of NLR, PLR, SII, and PNI were analyzed. Patient characteristics and changes in NLR, PLR, SII, and PNI before and after neoadjuvant chemotherapy (NAC) were compared between groups. Patients were divided into two groups according to the optimal diagnostic thresholds of the SII before treatment. Between-group differences in terms of neoadjuvant therapy efficacy and patient characteristics were evaluated. The pCR exhibited significantly lower ER (χ 2 = 10.227, P = 0.001), PR (χ 2 = 3.568, P = 0.049), pretreatment NLR (χ 2 = 24.930, P < 0.001), pretreatment PLR (χ 2 = 22.208, P < 0.001), pretreatment SII (χ 2 = 26.329, P < 0.001), and post-treatment PNI (P = 0.032), but higher HER-2 (χ 2 = 7.282, P = 0.007) and ΔNLR (P = 0.015) than the non-pCR group. ROC curve analysis revealed that the areas under the curve (AUC) of pretreatment SII, NLR, and PLR for predicting pCR of NAC for breast cancer were 0.827, 0.827, and 0.810, respectively, indicating a higher predictive value for response to NAC in patients with breast cancer. According to the Youden index, the optimal cut-off value of SII pretreatment was 403.20. Significant differences in age (χ 2 = 6.539, P = 0.01), ER (χ 2 = 4.783, P = 0.029), and HER-2 (χ 2 = 4.712, P = 0.030) were observed between high and low-SII groups. In conclusion, pretreatment NLR, PLR, and SII can be used as predictors of pCR in patients with breast cancer receiving neoadjuvant chemotherapy. The predictive value of pretreatment SII is higher, and patients with low SII are more likely to achieve pCR.

Discovery of an ene-reductase for initiating flavone and flavonol catabolism in gut bacteria.

Gut microbial transformations of flavonoids, an enormous class of polyphenolic compounds abundant in plant-based diets, are closely associated with human health. However, the enzymes that initiate the gut microbial metabolism of flavones and flavonols, the two most abundant groups of flavonoids, as well as their underlying molecular mechanisms of action remain unclear. Here, we discovered a flavone reductase (FLR) from the gut bacterium, Flavonifractor plautii ATCC 49531 (originally assigned as Clostridium orbiscindens DSM 6740), which specifically catalyses the hydrogenation of the C2-C3 double bond of flavones/flavonols and initiates their metabolism as a key step. Crystal structure analysis revealed the molecular basis for the distinct catalytic property of FLR. Notably, FLR and its widespread homologues represent a class of ene-reductases that has not been previously identified. Genetic and biochemical analyses further indicated the importance of FLR in gut microbial consumption of dietary and medicinal flavonoids, providing broader insight into gut microbial xenobiotic transformations and possible guidance for personalized nutrition and medicine.

Rapid Generation of Universal Synthetic Promoters for Controlled Gene Expression in Both Gas-Fermenting and Saccharolytic Clostridium Species.

Engineering solventogenic clostridia, a group of important industrial microorganisms, to realize their full potential in biorefinery application is still hindered by the absence of plentiful biological parts. Here, we developed an effective approach for rapid generation of a synthetic promoter library in solventogenic clostridia based on a dual-reporter system (catP-lacZ) and a widely used strong thl promoter. The yielded artificial promoters, spanning 2 orders of magnitude, comprised two modular components (the core promoter region and the spacer between RBS and the translation-initiating code), and the strongest promoter had an over 10-fold-higher activity than the original expression part Pthl. The test of these synthetic promoters in controlled expression of sadh and danK in saccharolytic C. acetobutylicum and gas-fermenting C. ljungdahlii, respectively, gave the expected phenotypes, and moreover, showed good correlation between promoter activities and phenotypic changes. The presented wide-strength-range promoters here will be useful for synthetic biology application in solventogenic clostridia.