Media development and process parameter optimization using statistical experimental designs for the production of nonribosomal peptides in Escherichia coli

BACKGROUND: Nonribosomal peptide synthases (NRPS) can synthesize functionally diverse bioactive peptides by incorporating nonproteinogenic amino acids, offering a rich source of new drug leads. The bacterium Escherichia coli is a well-characterized production host and a promising candidate for the synthesis of nonribosomal peptides, but only limited bioprocess engineering has been reported for such molecules. We therefore developed a medium and optimized process parameters using the design of experiments (DoE) approach. RESULTS: We found that glycerol is not suitable as a carbon source for rhabdopeptide production, at least for the NRPS used for this study. Alternative carbon sources from the tricarboxylic acid cycle achieved much higher yields. DoE was used to optimize the pH and temperature in a stirred-tank reactor, revealing that optimal growth and optimal production required substantially different conditions. CONCLUSIONS: We developed a chemically defined adapted M9 medium matching the performance of complex medium (lysogeny broth) in terms of product concentration. The maximum yield in the reactor under optimized conditions was 126 mg L-1, representing a 31-fold increase compared to the first shaking-flask experiments with M9 medium and glycerol as the carbon source. Conditions that promoted cell growth tended to inhibit NRPS productivity. The challenge was therefore to find a compromise between these factors as the basis for further process development.

Development of a real-time fluorescence quantitative PCR method for detection of FPGS mRNA expression in methotrexate enantiomer-resistant A549 cell lines and patients with leukemia / 中华检验医学杂志

Objective To establish a real-time fluorescence quantitative PCR method for detection of the different expression level of FPGS in methotrexate enantiomer-resistant A549 cell lines,and observe FPGS mRNA expression in patients with leukemia.Methods A real-time fluorescence quantitative PCR method for detection FPGS mRNA was established using SYBR Green Ⅰ as fluorescence and β-actin as reference.The method was evaluated by Ct,correlation coefficient,slope,repeatability curve,melting curve and amplification efficiency curve.The expression levels of FPGS gene in methotrexate enantiomer-resistant A549 cell lines and methotrexate resistant leukemia cells in bone marrow were detected by the method.Results The standard curves had a high linear relationship between cycle threshold and template concentration.The correlation coefficients of FPGS and β-actin were 0.996 8 and 0.998 7,and the slopes were -3.595 and -3.740,respectively.The inter-coefficient of variation was from 1.27% to 2.95%.The intra-coefficient of variation was 3.82%.The method was characterized with specific melting curve and similar amplification efficiency(slope was 0.021 7).The relative contents of FPGS mRNA were(3.51 ±0.66),(0.16 ±0.01) and(1.00 ±0.31) in L-(+)-MTX/A549 cells(L),D-(-)-MTX/A549 cells(D)and A549 parent cells,and there was statistically difference among the three groups(F = 64.45 ,P＜ 0.01)Statistical difference was observed between L and D(q =9.29,P＜0.01).After treated with MTX,the expression level of FPGS mRNA was(0.35 ± 0.04) in methotrexate resistant leukemia patients,compared with(1.00 ± 0.44) before treatment.Statistical difference was observed(t = 8.83 ,P＜ 0.01).Conclusions The real-time fluorescence quantitative PCR is suitable for the quantification of FPGS.The expression levels of FPGS in methotrexate resistant leukemia cells in bone marrow and drug resistant cells are different.Two enantiomer forms of methotrexate may play different roles in drug resistance mechanisms.

Detection of folylpolyglutamate synthetase in methotrexate enantiomer induced resistant A549 cells by capillary electrophoresis-laser-induced fluorescence / 中华检验医学杂志

Objective To establish a method for detection folylpolyglutamate syntbetase (FPGS),explore the change of FPGS in the drug-resistant A549 cells induced by methotrexate(MTX) enantiomer,and provide new tools to further investigate drug resistant mechanism. Methods A549 cell lines induced by L-( + )-MTX and D-( - )-MTX (25 μmol/L) were chosen to raise three cell lines as compared with MTX-sensitive cell line. Then FPGS were extracted for the CEIA-LIF and western blot was performed. After validation, FPGS antibodies were labeled by fluoreacein isothiocyanate (FITC) and produced a immune response with former-extracted FPGS. CELA-LIF can separate and detect labeled proteins according ruination time of the protein with different size and detect FPGS in drug resistant cell lines induced with L-(+)-MTX and D-(- )-MTX. The accuracy was evaluated as compared with western blot assay. Results The separation time of CEIA-LIF for labeled FPGS antibody and the immune complexes were7. 1 min and 8.9 min, and the resolving power was 4. 5. The process of protein separation and detection can be accomplished in less than 10 minutes. Western blot analysis showed there was no non-specific bands appears in the extract of these three cell lines after the freeze-thaw in liquid nitrogen. The minimum detection level in sensitive cell strains was 0. 68 mg/μl. The consent of FPGS in L-(+)-MTX and D-( - )-MTX induced cells were 46. 59% and 48. 36% compared with drug sensitive cell strains with CELA-LIF. Conclusions CELA-LIF was established in this experiment. It is efficient and sensitive for detecting of FPGS, which is similar to western blot method. The level of FPGS in L-( + )-MTX and D-( - )-MTX induced drug resistant cell lines is significantly lower, indicating the expression of FPGS is damaged.