Adaptive laboratory evolution for improved tolerance of vitamin K in Bacillus subtilis.

Menaquinone-7 (MK-7), a subtype of vitamin K2 (VK2), assumes crucial roles in coagulation function, calcium homeostasis, and respiratory chain transmission. The production of MK-7 via microbial fermentation boasts mild technological conditions and high biocompatibility. Nevertheless, the redox activity of MK-7 imposes constraints on its excessive accumulation in microorganisms. To address this predicament, an adaptive laboratory evolution (ALE) protocol was implemented in Bacillus subtilis BS011, utilizing vitamin K3 (VK3) as a structural analog of MK-7. The resulting strain, BS012, exhibited heightened tolerance to high VK3 concentrations and demonstrated substantial enhancements in biofilm formation and total antioxidant capacity (T-AOC) when compared to BS011. Furthermore, MK-7 production in BS012 exceeded that of BS011 by 76% and 22% under static and shaking cultivation conditions, respectively. The molecular basis underlying the superior performance of BS012 was elucidated through genome and transcriptome analyses, encompassing observations of alterations in cell morphology, variations in central carbon and nitrogen metabolism, spore formation, and antioxidant systems. In summation, ALE technology can notably enhance the tolerance of B. subtilis to VK and increase MK-7 production, thus offering a theoretical framework for the microbial fermentation production of other VK2 subtypes. Additionally, the evolved strain BS012 can be developed for integration into probiotic formulations within the food industry to maintain intestinal flora homeostasis, mitigate osteoporosis risk, and reduce the incidence of cardiovascular disease. KEY POINTS: • Bacillus subtilis was evolved for improved vitamin K tolerance and menaquinone-7 (MK-7) production • Evolved strains formed wrinkled biofilms and elongated almost twofold in length • Evolved strains induced sporulation to improve tolerance when carbon was limited.

Preparation of Cellulose-Based Flocculant and Its Application in the Enrichment of Vitamin K2 in Fermentation Supernatant.

Nutritional food supplements and pharmaceutical products produced with vitamin K2 as raw materials a very promising market in the global scope. The main production method of vitamin K2 is microbial fermentation, but approximately 50% of vitamin K2 synthesized by the main production strain Bacillus subtilis natto exists in extracellular form, which is not easy to separate and extract. In order to solve this problem, in this study, we synthesized a novel cellulose flocculant, MCC-g-LMA, by grafting reaction using microcrystalline cellulose (MCC) and lauryl methacrylate (LMA) as monomers, and ammonium persulfate as an initiator to flocculate VK2 from the fermentation supernatant. The flocculant was characterized by Fourier transform infrared spectroscopy (FTIR), elemental analysis, and scanning electron microscopy (SEM), and the grafting reaction was successful. When the flocculant dosage was 48.0 mg/L and pH was 5.0, the flocculation rate of the MCC-g-LMA on the fermentation supernatant reached 85.3%, and the enrichment rate of VK2 reached 90.0%. Furthermore, we explored the flocculation mechanism of VK2 by the MCC-g-LMA and speculated that the flocculation mechanism mainly included adsorption bridging, hydrophobic association and net trapping and sweep effect. In this study, the extraction method for trace high-value biological products in the fermentation supernatant was improved, which provided a method and theoretical basis for the efficient separation and purification of VK2 and other terpenoids.

Bottom-up synthetic biology approach for improving the efficiency of menaquinone-7 synthesis in Bacillus subtilis.

BACKGROUND: Menaquinone-7 (MK-7), which is associated with complex and tightly regulated pathways and redox imbalances, is produced at low titres in Bacillus subtilis. Synthetic biology provides a rational engineering principle for the transcriptional optimisation of key enzymes and the artificial creation of cofactor regeneration systems without regulatory interference. This holds great promise for alleviating pathway bottlenecks and improving the efficiency of carbon and energy utilisation. RESULTS: We used a bottom-up synthetic biology approach for the synthetic redesign of central carbon and to improve the adaptability between material and energy metabolism in MK-7 synthesis pathways. First, the rate-limiting enzymes, 1-deoxyxylulose-5-phosphate synthase (DXS), isopentenyl-diphosphate delta-isomerase (Fni), 1-deoxyxylulose-5-phosphate reductase (DXR), isochorismate synthase (MenF), and 3-deoxy-7-phosphoheptulonate synthase (AroA) in the MK-7 pathway were sequentially overexpressed. Promoter engineering and fusion tags were used to overexpress the key enzyme MenA, and the titre of MK-7 was 39.01 mg/L. Finally, after stoichiometric calculation and optimisation of the cofactor regeneration pathway, we constructed two NADPH regeneration systems, enhanced the endogenous cofactor regeneration pathway, and introduced a heterologous NADH kinase (Pos5P) to increase the availability of NADPH for MK-7 biosynthesis. The strain expressing pos5P was more efficient in converting NADH to NADPH and had excellent MK-7 synthesis ability. Following three Design-Build-Test-Learn cycles, the titre of MK-7 after flask fermentation reached 53.07 mg/L, which was 4.52 times that of B. subtilis 168. Additionally, the artificially constructed cofactor regeneration system reduced the amount of NADH-dependent by-product lactate in the fermentation broth by 9.15%. This resulted in decreased energy loss and improved carbon conversion. CONCLUSIONS: In summary, a "high-efficiency, low-carbon, cofactor-recycling" MK-7 synthetic strain was constructed, and the strategy used in this study can be generally applied for constructing high-efficiency synthesis platforms for other terpenoids, laying the foundation for the large-scale production of high-value MK-7 as well as terpenoids.

Engineering microbial consortia of Elizabethkingia meningoseptica and Escherichia coli strains for the biosynthesis of vitamin K2.

BACKGROUND: The study and application of microbial consortia are topics of interest in the fields of metabolic engineering and synthetic biology. In this study, we report the design and optimisation of Elizabethkingia meningoseptica and Escherichia coli co-culture, which bypass certain limitations found during the molecular modification of E. meningoseptica, such as resistance to many antibiotics and fewer available molecular tools. RESULTS: The octaprenyl pyrophosphate synthase from E. meningoseptica sp. F2 (EmOPPS) was expressed, purified, and identified in the present study. Then, owing to the low vitamin K2 production by E. coli or E. meningoseptica sp. F2 monoculture, we introduced the E. meningoseptica and E. coli co-culture strategy to improve vitamin K2 biosynthesis. We achieved production titres of 32 mg/L by introducing vitamin K2 synthesis-related genes from E. meningoseptica sp. F2 into E. coli, which were approximately three-fold more than the titre achieved with E. meningoseptica sp. F2 monoculture. This study establishes a foundation for further engineering of MK-n (n = 4, 5, 6, 7, 8) in a co-cultivation system of E. meningoseptica and E. coli. Finally, we analysed the surface morphology, esterase activity, and membrane permeability of these microbial consortia using scanning electron microscopy, confocal laser scanning microscopy, and flow cytometry, respectively. The results showed that the co-cultured bacteria were closely linked and that lipase activity and membrane permeability improved, which may be conducive to the exchange of substances between bacteria. CONCLUSIONS: Our results demonstrated that co-culture engineering can be a useful method in the broad field of metabolic engineering of strains with restricted molecular modifications.

Bioconversion of farnesol and 1,4-dihydroxy-2-naphthoate to menaquinone by an immobilized whole-cell biocatalyst using engineered Elizabethkingia meningoseptica.

Menaquinone (MK) has important applications in the pharmaceutical and food industries. To increase the production rate (QP) of MK-4, we developed a straightforward biotransformation method for MK-4 synthesis directly from its precursors 1,4-dihydroxy-2-naphthoate (DHNA) and farnesol using whole cells of genetically engineered Elizabethkingia meningoseptica. Results showed that MK-4 can be produced directly from farnesol and DHNA using both free and immobilized FM-D198 cells. MK-4 yield peaked at 29.85 ± 0.36 mg/L in the organic phase and 24.08 ± 0.33 mg/g DCW after 12 h of bioconversion using free cells in a two-phase conversion system. MK-4 yield reached 26.34 ± 1.35 mg/L and 17.44 ± 1.05 mg/g DCW after 8 h using immobilized cells. Although this yield was lower than that using free cells, immobilized cells can be re-used for MK-4 production via repeated-batch culture. After ten batch cultures, efficient MK-4 production was maintained at a yield of more than 20 mg/L. After optimizing the catalysis system, the MK-4 yield reached 26.91 ± 1.27 mg/L using the immobilized cells and had molar conversion rates of 58.56 and 76.90% for DHNA and farnesol, respectively.

The change of the state of cell membrane can enhance the synthesis of menaquinone in Escherichia coli.

Menaquinone (MK) was an attractive membrane-bound intracellular chemical. To enhance its production, we tried to find the relationship between its synthesis and the state of cell membrane in producing strain. Due to non-ionic surfactant-polyoxyethylene oleyl ether (POE) and plant oil-cedar wood oil (CWO) can typically increase extracellular secretion and intracellular synthesis of MK respectively, the effect of these two substances on cell morphology, physical properties of cell membrane was investigated. Finally, two engineering strains were constructed to verify whether the state of cell membrane can enhance MK synthesis. The result showed that the edge of cells was broken when POE added in the medium. Other physical properties such as total fatty acid content decreased by 40.7% and the ratio of saturated fatty acids to unsaturated fatty acids decreased from 1.58 ± 0.05 to 1.31 ± 0.04. Meanwhile, cell membrane leakage was enhanced from 7.14 to 64.31%. Different from POE group, cell membrane was intact in CWO group. Moreover, the ratio of saturated fatty acids to unsaturated fatty acids increased from 1.58 ± 0.05 to 1.78 ± 0.04 and the average lipid length decreased from 16.05 ± 0.08 to 15.99 ± 0.10. Two constructed strains, especially Escherichia coli DH5α FatB, exhibited strong MK secretion ability and the extracellular MK reached 10.71 ± 0.19 mg/L. An understanding of these functionary mechanisms could not only provide a new idea for the synthesis of MK, but also provide a reference to increase the yield of intracellular membrane-bound metabolites.

Oncogenic role of Skp2 and p27Kip1 in intraductal proliferative lesions of the breast / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate whether the connection of p27(Kip1) to S-phase kinase-associated protein 2 (Skp2) plays an oncogenic role in intraductal proliferative lesions of the breast.</p><p><b>METHODS</b>Here we investigated the mechanism involved in association of Skp2’s degradation of p27(Kip1) with the breast carcinogenesis by immunohistochemical method through detection of Skp2 and p27(Kip1) protein levels in 120 paraffin-embedded tissues of intraductal proliferative lesions including usual ductal hyperplasia (UDH, n=30), atypical ductal hyperplasia (n=30), flat epithelial atypia (FEA, n=30), and ductal carcinoma in situ (DCIS, n=30). Moreover, the expression status of Skp2 and p27(Kip1) in 30 cases of the normal breast paraffin-embedded tissues were explored.</p><p><b>RESULTS</b>The DCIS group was with the highest Skp2 level and the lowest p27(Kip1) level, and the UDH group was with the lowest Skp2 level and the highest p27(Kip1) level.Both Skp2 and p27(Kip1) levels in the DCIS group were significantly different from those in the UDH group (all P<0.01).The levels of Skp2 and p27(Kip1) in the FEA group were significantly different from both the DCIS and UDH groups (all P<0.05).p27(Kip1) was negatively correlated with Skp2 in both the UDH group (r=-0.629, P=0.026) and DCIS group (r=-0.893, P=0.000).</p><p><b>CONCLUSION</b>Overexpression of Skp2 might be the mechanism underlying p27(Kip1) over degradation.</p>

Analysis of the progression of intraductal proliferative lesions in the breast by PCR-based clonal assay.

PURPOSE: To analyze the progression in patients with a morphological diagnosis of intraductal proliferative lesions by PCR-based clonal assay. MATERIALS AND METHODS: An X-chromosome inactivation assay was applied to explore clonal relationships in human intraductal proliferative lesions of the breast. Four groups samples, including 40 cases of usual ductal hyperplasia (UDH), 40 cases of atypical ductal hyperplasia (ADH), 29 cases of flat epithelia atypia (FEA), and 40 cases of ductal carcinoma in situ (DCIS) were selected for analysis. Thirty specimens of normal breast tissue were used as a control group. Microdissection was performed to collect the tissue samples for extraction of genomic DNA from paraffin-embedded tissues. The DNA was subjected to PCR amplification of the CAG repeats in androgen receptor (AR) gene exon I with and without prior digestion of methylation-sensitive restriction enzyme HhaI. Gel electrophoresis was used to detect the clonal nature of these four groups samples. RESULTS: The clonal analysis confirmed monoclonality in all informative samples of DCIS cells. Normal tissues and the majority (97.1%) of UDH were shown to be polyclonal. Monoclonality was revealed in 20/39 (51.3%) cases of ADH. Among 26 cases of FEA, 20 were shown to be polyclonal, while six displayed monoclonal alterations which accounted for 23.1%. CONCLUSION: These findings reinforce recent suggestions that clonal analysis with AR gene polymerase chain reaction may be used to define the genetic relationships among the human tumor and the breast intraductal proliferative lesions. Furthermore, our observations demonstrate nearly a half ADH and the smaller part of FEA have clonal alterations, which may be neoplastic lesions. This method would shed light on genetic abnormalities that play a role in early tumorigenesis of the breast, and thus might be an adjunct in predicting the probability of breast tumor occurrence and in guiding the management of these cases.

[Clonality of the peripheral papilloma and cancerous cells of breast].

OBJECTIVE: To study the clonality status of peripheral papilloma (peri-MP), ductal carcinoma in situ (DCIS), and normal tissue of the breast using an assay based on inactivation mosaicism of the length-polymorphic X-chromosomes at the androgen receptor (AR) locus and to explore a reliable way to distinguish the benign and malignant (or pre-malignant) cases judged morphologically. METHODS: Specimens of breast tissues were obtained from 26 cases of peri-PM, 25 cases of peri-PM with atypical ductal hyperplasia (ADH), and 27 cases pf DCIS, 16 cases of developed canceration, and 20 normal women. DNA was extracted and amplified via nested-PCR with or without previous digestion by the methylation-sensitive restriction endonuclease Hha I. The products were resolved on denaturing polyacrylamide gels and visualized through silver staining. The clonality of these samples was analyzed by showing the lanes. RESULTS: Loss of polymorphism at the AR locus was found in all the cases with DCIS and in 10 cases (10/25, 40.0%) of peri-PM with ADH, indicating the monoclonality of the tumor. Twenty-four cases (92.3%) of the 26 cases with peri-PM and the 20 specimens of normal tissue were shown to be polyclonal. In the 16 cases of developed canceration identical X chromosome inactivation (monoclonal alterations) was observed in the cancer focus, parts of peri-PM with ADH, and the part of DCIS. CONCLUSION: Normal breast tissue and peri-PM show polyclonality and the peri-PM with ADH shows monoclonality. Clonality analysis may be a useful modality to screen high-risk cases from precancerous lesions or to distinguish between the benign hyperplasia and early carcinoma.

[Expression of alpha-tubulin and gamma-tubulin in premalignant lesion and carcinoma of breast and the significance thereof].

OBJECTIVE: To investigate the expression of alpha-tubulin and gamma-tubulin, 2 kinds of centrosome proteins, in premalignant lesion and carcinoma of breast and the significance thereof. METHODS: Forty specimens of premalignant lesions of breast, 40 specimens of infiltrating ductal carcinoma of breast, 40 specimens of intraductal carcinoma (IDC), and 30 specimens of normal breast tissues were obtained during operation. Immunohistochemistry was used to analyze the protein expression of alpha-tubulin and gamma-tubulin, and the percentages of ki67 positive cells. Western blotting was used to examine the mRNA expression of alpha-tubulin and gamma-tubulin. RESULTS: The protein and mRNA expression values of alpha-tubulin and gamma-tubulin in breast carcinoma were higher than those in the premalignant lesions and normal breast tissues with significant differences between the premalignant lesions and normal breast torques and without significant differences between infiltrating ductal carcinoma of breast and IDC. The ki67 positive rates of the infiltrating ductal carcinoma of breast group, IDC group, premalignant lesion group, and normal breast tissues group were 16.0%, 37.0%, 53.6%, and 67.8% (P = 0.001). A positive correlation existed between the expression of alpha-tubulin and the expression of gamma-tubulin in the same case and the same group (all P = 0.00) and there was no significant correlation between the expression of alpha-tubulin and the expression of gamma-tubulin in the same case and the same group (all P > 0.05). Both the expression of alpha-tubulin and the expression of gamma-tubulin were significantly associated with the prognosis. CONCLUSIONS: Centrosome protein is one of the distinct phenotypes of breast cancer cells. Aberration of centrosome proteins may be used to screen high risk cases of breast carcinoma and to estimate the prognosis.