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.

[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.