Construction and optimization of Escherichia coli for producing rhamnolipid biosurfactant / 生物工程学报

Rhamnolipid biosurfactant is mainly produced by Pseudomonas aeruginosa that is the opportunistic pathogenic strain and not suitable for future industrial development. In order to develop a relatively safe microbial strain for the production of rhamnolipid biosurfactant, we constructed engineered Escherichia coli strains for rhamnolipid production by expressing different copy numbers of rhamnosyltransferase (rhlAB) gene with the constitutive synthetic promoters of different strengths in E. coli ATCC 8739. We further studied the combinatorial regulation of rhlAB gene and rhaBDAC gene cluster for dTDP-1-rhamnose biosynthesis with different synthetic promoters, and obtained the best engineered strain-E. coli TIB-RAB226. Through the optimization of culture temperature, the titer of rhamnolipd reached 124.3 mg/L, 1.17 fold higher than that under the original condition. Fed-batch fermentation further improved the production of rhamnolipid and the titer reached the highest 209.2 mg/L within 12 h. High performance liquid chromatography-mass spectrometry (LC-MS) analysis showed that there are total 5 mono-rhamnolipid congeners with different nuclear mass ratio and relative abundance. This study laid foundation for heterologous biosynthesis of rhanomilipd.

Pathological changes of human hepatocellular carcinoma after continuous passaging in nude mice / 中国肿瘤生物治疗杂志

Objective: To investigate the pathological changes of human hepatocellular carcinoma (HCC) after continuous passaging in nude mice. Methods: The mice model of HCC SMMU-LTMN were continuously observed for 20 years (1987-2007) . The subcutaneously transplanted carcinoma had been passaged for 228 generations. The pathological data of abdominally transplanted HCC, orthotopically transplanted HCC in nude mice, and orthotopically transplanted HCC in NOD-SCID mice were recorded. The pathological studies were conducted by light microscope, electron microscope, image analysis, chromosomal analysis, and measurement of alpha-fetoprotein (AFP) in peripheral blood. Results: (1) The local invasion and metastasis of of tumors were present in all the above 4 models for a long time. The local invasion rate and the pulmonary metastasis rate of subcutaneously transplanted tumors were 59.70% (40/67) and 37.10% (23/62), respectively. The pulmonary metastasis rate of abdominally transplanted tumors was 59.02%(36/61). The intra-hepatic and pulmonary metastasis rate of the othotopically transplanted tumors were 18.18%(4/22) and 31.82% (7/22), respectively. The pulmonary metastasis rate of HCC in NOD-SCID mice was 53.85%. (2) The tissue structure and the differentiation of the 10th generation tumor cells was similar to those of primary HCC, with grade 2 differentiation and coarse trabecular pattern as the main characteristics. From the 11th generation to the 228th generation, the main characteristics of tumor cells were grade 3 differentiation and lump pattern. Electron microscope also showed worse differentiation. (3)The AFP level was 92 500 ?g/L in cells before the 32th generation; it decreased to 6 729?g/L from the 33th-130th generation cells; and the level of the 220th generation was 1 000-5 000 ?g/L.(4)The DNA contents had a wide distribution (from 2c to 6c) in abdominally transplanted tumors and the pulmonary metastatic tumors; the mean DNA index in the former tumors (2.60?0.20) was wider than the that in the latter (2.10?0.26) . (5)From the 55th generation to 206th generation, it was found that tumor cells had integrated into the chromosome of the nude mice. Conclusion: The subcutaneously transplanted HCC in nude mice can be stably expressed for 20 years, with no change in the local invasion and metastasis ability of HCC. The differentiation of the tumor cells worsenes and the AFP level is decreased in the blood; some chromosome of tumor cells integrate into the chromosome of nude mice, which may be related to the internal environment of nude mice and the multi-potential differentiation of the tumor cells.