Qinghai Liu

Metabolic Adaptation of Mycobacterium neoaurum ATCC 25795 in the Catabolism of Sterols for Producing Important Steroid Intermediates

To understand the adaptation of Mycobacterium neoaurum ATCC25795 ( Mn) in sterol catabolism and steroid production, we used integrated transcriptome and proteome analysis to identify the biochemical pathways utilized in this process. Metabolic alterations during sterol catabolism center on propionyl-CoA pools. Generally, enhanced pathways for metabolizing propionyl-CoA were found in Mn, which were tightly coordinated with cell-envelope biosynthesis. The cells responded to sterol substrates and toxic steroid products by changing the composition of the cell envelope. This adaptive mechanism allowed Mn to use minimally water-soluble sterol as a carbon source. Several putative efflux proteins were found to be induced in Mn. They probably transported products to the extracellular environment, protecting the cells against high intracellular levels of toxic intermediates, inhibition of which also influenced sterol uptake. The work provided various targets for rational engineering of robust Mn with powerful sterol-uptake capacity and strong tolerance to toxic products for the steroid industry.

Integrated Transcriptome and Proteome Studies Reveal the Underlying Mechanisms for Sterol Catabolism and Steroid Production in Mycobacterium neoaurum

Integrated transcriptome and proteome studies were performed to investigate sterol biotransformation in wild-type Mycobacterium neoaurum ATCC 25795 ( Mn) and the mutant strains producing steroid intermediates. Transcriptome and proteome studies indicated that several metabolic activities were noticeably dynamic, including cholesterol degradation, central carbon metabolism, cell envelope biosynthesis, glycerol metabolism, and transport. Interestingly, a poor overall correlation between mRNA and translation profiles was found, which might contribute to the metabolic adaptation in cholesterol catabolism. A gene cluster covering 111 genes was discovered to encode for cholesterol catabolism in Mn. Generally, transcription and/or translation of the genes in KstR1 regulon was upregulated, and the induction of genes in KstR2 regulon was not as significant as that of KstR1 regulon. Several induced genes showing potential roles for cholesterol catabolism were found. Further identification of these genes and investigation of the correlation among key metabolic activities could help for the development of efficient steroid-producing strains.

Preparation of Recombinant HIV-TATm-Survivin (T34A) Protein and Its Proapoptosis Activity to Four Cancer Cell Lines in vitro

Abstract In this study, the cDNA encoding survivin was cloned by RT-PCR obtained from human breast cancer cell lines, B-Cap37. After the mutation of survivin (T34A) by site-directed mutagenesis, an expression vector of pRSET-B-HIV-TATm-Survivin (T34A) was constructed by PCR. Subsequently, the resultant plasmid was transformed into E. coli BL21 (DE3). The recombinant HIV-TATm-Survivin (T34A) protein was expressed efficiently by inducing IPTG at a concentration of 0.5 mmol/L, reaching a yield of 650 mg/L (as inclusion body) in fermentation culture. The inclusion bodies were solubilized, refolded, and purified by ion exchange chromatography and size-exclusion chromatography to a purity of 96 %. Remarkable effects of the purified recombinant HIV- TATm-Survivin (T34A) on the morphology of cell line SW1990 and B-Cap37 were observed after being administrated for 4 h. MTT assay showed that the recombinant HIV-TATm-Survivin (T34A) protein could restrain the cell proliferation of SW1990, B-Cap37, and SSMC-7721, in vitro. Apoptosis rate and cell circle of SW1990 and B-Cap37 that were treated with the target protein (final concentration 60 μg/mL) were detected with flow cytometry. The results revealed that the apoptosis rate of SW1990 and B-Cap37 were 25.6 % and 19.3 %, respectively. More than 65 % of cancer cells were arrested at the G1 phase. The study suggested that TATm-Survivin (T34A) protein was a hopeful protein drug for the treatment of cancer by facilitating apoptosis of cancer cells.